Biochemistry, Genetics and Molecular Biology › Molecular Biology

DNA Repair Mechanisms

Works Count: 95899

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Description

This cluster of papers focuses on the molecular mechanisms of the DNA damage response, including DNA repair pathways, genomic instability, the role of ATM and ATR kinases, homologous recombination, cell cycle checkpoints, and their implications in cancer biology. It also explores the involvement of histone H2AX and chromatin in maintaining genome stability.

Keywords

DNA Damage; Repair; Genomic Instability; ATM and ATR Kinases; Homologous Recombination; Cell Cycle Checkpoints; Cancer; Histone H2AX; Chromatin; Genome Stability

8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-T and A-C substitutions.

1992-01-01

Keith C. Cheng , D S Cahill , Hiroshi Kasai +2 more

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity … Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G→T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A→C substitutions, and two were G→T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G→T substitutions and misincorporation of oh8Gua as substrate causing A→C substitutions, both caused by oh8Gua.A mispairs.

DNA double-strand breaks: signaling, repair and the cancer connection

2001-03-01

Kum Kum Khanna , Stephen P. Jackson

Genomic instability — an evolving hallmark of cancer

2010-02-23

Simona Negrini , Vassilis G. Gorgoulis , Thanos D. Halazonetis

The DNA damage response: putting checkpoints in perspective

2000-11-01

Bin‐Bing S. Zhou , Stephen J. Elledge

Cell-cycle checkpoints and cancer

2004-11-01

Michael B. Kastan , Jiří Bártek

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy

2005-04-01

Hannah Farmer , Nuala McCabe , Christopher J. Lord +7 more

DNA Damage, Aging, and Cancer

2009-10-07

Jan H.J. Hoeijmakers

This review gives an account of how the cell repairs DNA damage and presents evidence that DNA damage contributes to aging and cancer, with the outcome dependent on the type … This review gives an account of how the cell repairs DNA damage and presents evidence that DNA damage contributes to aging and cancer, with the outcome dependent on the type and number of lesions in DNA. Examples of accelerated aging syndromes associated with defects in DNA repair mechanisms are contrasted with cancers.

Defective Repair Replication of DNA in Xeroderma Pigmentosum

1968-05-01

James E. Cleaver

DNA repair and mutagenesis

1996-02-01

D. Bootsma , Jan H.J. Hoeijmakers

ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks

2001-11-01

Sandeep Burma , Benjamin P. Chen , Michael Murphy +2 more

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although … A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. double-strand break ionizing radiation phosphatidylinositol mouse embryonic fibroblasts DNA double-strand breaks (DSBs)1 are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination (1Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1867) Google Scholar). A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar) resulting in discrete γ-H2AX (phosphorylated-H2AX) foci at the DNA damage sites (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). In experiments involving the use of “laser scissors” to introduce breaks into living cells, γ-H2AX foci localized specifically with the laser path through the cell nuclei clearly demonstrating that H2AX phosphorylation is specific to the sites of DNA damage (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells (6Chen H.T. Bhandoola A. Difilippantonio M.J. Zhu J. Brown M.J. Tai X. Rogakou E.P. Brotz T.M. Bonner W.M. Ried T. Nussenzweig A. Science (Wash. D. C.). 2000; 290: 1962-1964Crossref PubMed Scopus (283) Google Scholar) and meiotic recombination in mice (7Mahadevaiah S.K. Turner J.M.A. Baudat F. Rogakou E.P. de Boer P. Blanco-Rodriguez J. Jasin M. Keeney S. Bonner W.M. Burgoyne P.S. Nat. Genet. 2001; 27: 271-276Crossref PubMed Scopus (685) Google Scholar). Phosphorylation of yeast H2A at serine 129 (homologous to serine 139 of mammalian H2AX) causes chromatin decondensation and is required for efficient DNA double-strand break repair (8Downs J.A. Lowndes N.F. Jackson S.P. Nature. 2000; 408: 1001-1004Crossref PubMed Scopus (529) Google Scholar). In mammals, phosphorylation of H2AX appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 9Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (396) Google Scholar). As H2AX phosphorylation plays a very early and important role in the cellular response to DNA double-strand breaks, it is important to specifically identify the kinase(s) involved in this event. Members of the PI 3-kinase family, including DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), are involved in the responses of mammalian cells to DSBs (10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). γ-H2AX focus formation is inhibited by the PI 3-kinase inhibitor wortmannin, and H2AX phosphorylation is reduced in the DNA-PK-deficient human cell line M059J (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). This led to the conclusion that DNA-PK and at least one other kinase, possibly ATM and/or ATR, can phosphorylate H2AX upon DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). To unambiguously define the roles of ATM and DNA-PK in H2AX phosphorylation, we utilized cells derived from knockout mice for ATM or DNA-PKcs (the catalytic subunit of DNA-PK). We observed normal H2AX phosphorylation and γ-H2AX focus formation in irradiated fibroblasts derived from wild type or DNA-PKcs−/− mice. In contrast, H2AX phosphorylation and γ-H2AX focus formation were strikingly reduced to near background levels in fibroblasts from Atm−/− mice. Ectopic expression of ATM in Atm−/− cells restored H2AX phosphorylation. Moreover, we show that immunoprecipitated ATM can phosphorylate recombinant H2AX in vitro. These results indicate that ATM, not DNA-PK, is the major kinase responsible for modifying H2AX upon irradiation. The minimal H2AX phosphorylation in Atm−/− cells could be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. Spontaneously immortalized mouse fibroblasts, derived from wild type, DNA-PKcs−/− (12Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (158) Google Scholar), or Atm−/− mice (13Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Genes Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (736) Google Scholar), were maintained in a humidified atmosphere with 5% CO2 in α-minimum Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Mouse embryonic fibroblasts (MEFs) were isolated from 13.5-day-old embryos and maintained in α-minimum Eagle’s medium supplemented with 15% fetal calf serum. Cells were grown to about 70% confluence and irradiated with x-rays (300-kV, 12-mA, 0.5-mm Cu) at the rate of 5.5 gray/min to achieve a cumulative dose of 10 gray for all experiments unless otherwise mentioned. Cells were UV-irradiated at the rate of 0.15 J/m2/s to achieve a cumulative dose of 10 J/m2. Cells were harvested after 30 min, except in the case of time courses where they were harvested at time points ranging from 5 min to 8 h. Drug treatment of cells was carried out by the addition of the following DNA-damaging agents to the culture media for 1 h at the indicated concentrations: neocarzinostatin (0.2 μg/ml), bleomycin (50 μg/ml), etoposide (30 μg/ml), methyl methanesulfonate (50 μg/ml), and hydroxyurea (1 mm). Anti-γ-H2AX antibody was generated against a synthetic peptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 as described before (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar). SDS extracts for Western blotting were prepared from mock-irradiated or irradiated cells as described previously (14D'Anna J.A. Valdez J.G. Habbersett R.C. Crissman H.A. Radiat. Res. 1997; 148: 260-271Crossref PubMed Scopus (31) Google Scholar). The antibodies used for Western blotting are anti-γ-H2AX, anti-H2A (H-124; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). Transient transfection of exponentially growing Atm−/− spontaneously immortalized fibroblasts with the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was carried out using Superfect transfection reagent (Qiagen Inc., Valencia, CA) as per the manufacturer’s protocols. Immediately after transfection, cells were induced for ATM expression with 5 μm CdCl2 for 16 h and then mock-irradiated or irradiated as described above. ATM immunoprecipitations were carried out as described (17Ziv Y. Banin S. Lim D.S. Canman C.E. Kastan M.B. Shiloh Y. Chan D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. Methods Mol. Biol. 2000; 99: 99-108PubMed Google Scholar). Approximately 1 × 107spontaneously immortalized mouse fibroblasts were grown to 70% confluence, mock-irradiated or irradiated, harvested after 30 min, and lysed in fresh cold lysis buffer containing protease and phosphatase inhibitors. The lysate was cleared by centrifugation, and the supernatant was incubated with 10 μg of anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar) for 2 h at 4 °C followed by incubation with protein A/G-Sepharose beads for an additional 2 h. The beads were washed repeatedly with lysis buffer, once with high salt buffer, and twice with kinase buffer. The beads were then incubated in a kinase mix (20 μl of kinase buffer, 500 ng of recombinant H2AX (purified from bacteria), 2 μl of 100 μm ATP, and 10 μCi of γ[32P]ATP) at 30 °C for 10 min. After SDS-polyacrylamide gel electrophoresis, the reaction products were visualized by autoradiography. Spontaneously immortalized fibroblasts were grown on chamber slides to about 70% confluence and then mock-irradiated or irradiated and incubated for 30 min. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized for 10 min in 0.2% Triton X-100, and blocked in 10% normal goat serum for 1 h at room temperature. The slides were incubated with anti-γ-H2AX antibody for 1 h, washed in phosphate-buffered saline, and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were washed in phosphate-buffered saline and mounted using Vectashield mounting medium with 4,6 diamidino-2-phenylindole (Vector Laboratory, Burlingame, CA). Fluorescence images were captured using an Olympus BH2 epifluorescent microscope equipped with a CCD camera and Cytovision software (Applied Imaging, Santa Clara, CA). To allow direct comparisons, all the cells were irradiated and processed simultaneously, and all the images were obtained using the same parameters (brightness, contrast, etc.). To examine H2AX phosphorylation in mouse cells, a rabbit polyclonal antibody (anti-γ-H2AX) was generated against a synthetic phosphorylated polypeptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 (CKATQAS(PO4)QEY). The purified anti-γ-H2AX antibody reacted specifically with the immunizing polypeptide (phosphorylated at serine 139) but not with the unphosphorylated peptide (CKATQASQEY) (Fig. 1 a). Thus, the anti-γ-H2AX antibody is immunoreactive only with H2AX specifically phosphorylated at serine 139. Spontaneously immortalized wild type mouse fibroblasts were mock-irradiated or irradiated with x-rays and harvested after 30 min, and H2AX phosphorylation was analyzed by Western blotting of SDS extracts with anti-γ-H2AX antibody. We observed significant phosphorylation of histone H2AX in response to ionizing radiation (Fig. 1 b). The observed phosphorylation was specific to serine 139 as no signal was detected in irradiated samples when the immunizing polypeptide (phosphorylated at serine 139) was used as competitor in Western blotting (data not shown). Significant phosphorylation of H2AX was also observed after treatment of cells with the DSB-inducing agents neocarzinostatin, bleomycin, and etoposide. In contrast, there was no increase in γ-H2AX formation when these cells were irradiated with UV rays or treated with the DNA-alkylating agent methyl methanesulfonate confirming that H2AX is phosphorylated at serine 139 specifically in response to DNA double-strand breaks. Low levels of H2AX phosphorylation were also observed in cells treated with the DNA replication inhibitor hydroxyurea. This is probably because cells treated with hydroxyurea accumulate DSBs because of replication fork collapse (18Saintigny Y. Delacote F. Vares G. Petitot F. Lambert S. Averbeck D. Lopez B.S. EMBO J. 2001; 20: 3861-3870Crossref PubMed Scopus (259) Google Scholar, 19Skog S. Heiden T. Eriksson S. Wallström B. Tribukait B. Anticancer Drugs. 1992; 3: 379-386Crossref PubMed Scopus (13) Google Scholar). As the PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in H2AX phosphorylation (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar,10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar), we wanted to determine which of these three kinases played a major role in the process. The fungal PI 3-kinase inhibitor wortmannin inhibits the kinase activities of ATM and DNA-PK in intact cells with half-maximal inhibition at concentrations of about 5 μm(20Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar). The kinase activity of ATR is significantly more resistant to this drug with half-maximal inhibition at concentrations higher than 100 μm. Spontaneously immortalized wild type mouse fibroblasts were treated with increasing concentrations of wortmannin for 30 min, irradiated with x-rays, harvested after 30 min, and analyzed by Western blotting. We found that H2AX phosphorylation was inhibited by low concentrations of wortmannin (1–10 μm) indicating that ATM and/or DNA-PK, but not ATR, is involved in this process (Fig. 1 c). Spontaneously immortalized fibroblasts from wild type, DNA-PKcs−/−, or Atm−/− mice were mock-irradiated or irradiated, harvested at time points ranging from 5 min to 8 h, and assayed for H2AX phosphorylation by Western blotting. H2AX phosphorylation in both wild type and DNA-PKcs−/− cells occurred very rapidly (within 5 min) and lasted for about 2 h, with maximum levels of phosphorylation observed at 30 min (Fig. 2 a). In striking contrast, we observed minimal H2AX phosphorylation in irradiated Atm−/− cells. Although we observed robust H2AX phosphorylation in DNA-PKcs−/− cells at 30 min post-irradiation, γ-H2AX formation in Atm−/− cells was reproducibly reduced to about 5% of that in wild type cells (Fig. 2 b) indicating that ATM is the major kinase responsible for H2AX phosphorylation upon DNA damage. Atm−/− fibroblasts were treated with increasing concentrations of wortmannin for 30 min, irradiated with x-rays, harvested after 30 min, and analyzed by Western blotting. We found that the minimal H2AX phosphorylation in Atm−/− cells was completely abolished by low concentrations of wortmannin (1–10 μm) (Fig. 2 c). As ATR is inhibited by high concentrations of wortmannin (>100 μm) (20Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar), our results suggest that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. It is possible that other mutations in the Atm−/− cell line used could also be responsible for the lack of H2AX phosphorylation in these cells. We, therefore, examined H2AX phosphorylation in a panel of isogenic, early passage (p2 or p3) ATM+/+ or −/− MEFs. Normal H2AX phosphorylation was observed in irradiated Atm+/+ MEFs (Fig. 3 a, upper panel). In contrast, very low levels of γ-H2AX formation was observed in two independent Atm−/− MEFs confirming that ATM is required for H2AX phosphorylation in response to IR. No significant difference in H2AX phosphorylation was observed between irradiated DNA-PKcs+/− and −/− MEFs (Fig. 3 a, lower panel). To confirm that ATM is required in vivo for H2AX phosphorylation, the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was transiently transfected into Atm−/− spontaneously immortalized fibroblasts. The ectopic expression of ATM in the transfected cells resulted in restoration of H2AX phosphorylation upon irradiation (Fig. 3 b, compare lanes 2 and 4). On the other hand, cells transfected with the vector alone showed no increase in γ-H2AX formation (Fig. 3 b, compare lanes 2and 6). The correlation between ATM expression and H2AX phosphorylation establishes that ATM is required in vivo for γ-H2AX formation in response to ionizing radiation. To determine whether ATM can directly phosphorylate H2AX in vitro, ATM was immunoprecipitated from spontaneously immortalized wild type fibroblasts using an anti-ATM monoclonal antibody raised against a peptide representing positions 1967–1988 of murine ATM (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). The immunoprecipitated ATM efficiently phosphorylated recombinant H2AX in vitro (Fig. 3 c, lane 1). Furthermore, irradiation of cells resulted in a significant increase in H2AX phosphorylation (Fig. 3 c, lane 2). Essentially no ATM protein or kinase activity was detected when immunoprecipitation was performed with normal mouse IgG or from Atm−/− fibroblasts (Fig. 3 c, lanes 3–6). The in vitro phosphorylation of H2AX by ATM suggests that ATM could directly phosphorylate H2AX within the cell in response to DNA damage. H2AX phosphorylation in response to DNA damage results in the formation of discrete γ-H2AX foci at the sites of DNA double-strand breaks (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). To determine the status of γ-H2AX focus formation in wild type, DNA-PKcs−/−, and Atm−/− spontaneously immortalized fibroblasts, these cells were irradiated and allowed to recover for 30 min before fixation and immunostaining with anti-γ-H2AX antibody. We observed robust γ-H2AX focus formation upon irradiation of both wild type and DNA-PKcs−/− cells (Fig. 4). In striking contrast, focus formation was very poor in Atm−/− cells confirming that ATM is required for γ-H2AX focus formation at the sites of DSBs. Histone H2AX is rapidly phosphorylated at serine 139 in response to DNA double-strand breaks (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar). The PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in this process (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). Although the substrate specificities of these kinases are overlapping in vitro, they have clearly distinct functions in vivo(10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). For example, whereas ATM phosphorylates p53, Chk2, and Nbs1 leading to cell cycle arrest, DNA-PK is not required for any of these processes (21Burma S. Kurimasa A. Xie G. Taya Y. Araki R. Abe M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; 274: 17139-17143Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (652) Google Scholar). On the other hand, DNA-PK, unlike ATM, may be involved in the recruitment of XRCC4 and DNA ligase IV to the sites of DSBs (23Smith G.C. Jackson S.P. Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (764) Google Scholar). It is therefore important to definitively delineate the roles of these kinases in the phosphorylation of H2AX. We demonstrate that ATM can phosphorylate H2AX in vitro and that H2AX phosphorylation and γ-H2AX focus formation are severely compromised in Atm−/− cells. Ectopic expression of ATM corrects this defect. In contrast, these functions are normal in DNA-PKcs−/− cells. Interestingly, DNA-PK, but not ATR, may be responsible for the minimal levels of H2AX phosphorylation in Atm−/− cells as this can be abolished by low concentrations of wortmannin. We also find that immunoprecipitated ATM can directly interact with recombinant H2AX in vitro, 2Sandeep Burma and David J. Chen, unpublished results. and experiments will be performed to examine complex formation between H2AX and ATM in vivo. Our results establish that ATM is the major kinase responsible for histone H2AX phosphorylation in response to DNA double-strand breaks in murine fibroblasts. The reduced H2AX phosphorylation reported in M059J cells (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar) could, therefore, be because of the low levels of ATM in these cells (24Hoppe B.S. Jensen R.B. Kirchgessner C.U. Radiat. Res. 2000; 153: 125-130Crossref PubMed Scopus (61) Google Scholar, 25Gately D.P. Hittle J.C. Chan G.K. Yen T.J. Mol. Biol. Cell. 1998; 9: 2361-2374Crossref PubMed Scopus (163) Google Scholar, 26Chan D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. Int. J. Radiat. Biol. 1998; 74: 217-224Crossref PubMed Scopus (66) Google Scholar) rather than because of the absence of DNA-PK. ATM plays a crucial role in the rapid induction of multiple signaling pathways in response to DSBs, leading to repair of DNA damage, activation of cell cycle checkpoints, and cellular stress responses (27Rotman G. Shiloh Y. Oncogene. 1999; 18: 6135-6144Crossref PubMed Scopus (230) Google Scholar). As γ-H2AX focus formation is a very early event occurring within seconds of DNA damage infliction (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar), our results indicate that ATM is one of the earliest kinases to be activated in the cellular response to DNA double-strand breaks. Supporting the important role of ATM in damage-induced chromatin modification is a report indicating that ATM is also required for the IR-induced transient dephosphorylation of histone H1, which is thought to contribute to chromatin decondensation (28Guo C.Y. Wang Y. Brautigan D.L. Larner J.M. J. Biol. Chem. 1999; 274: 18715-18720Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Our results are consistent with a recent report indicating that a fraction of nuclear ATM co-localizes with γ-H2AX at the sites of DSBs in response to DNA damage (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). A very striking correlation was reported between the kinetics of appearance and dissolution of γ-H2AX foci and DNA-bound ATM aggregates. This suggests that DNA localization by ATM and H2AX phosphorylation occur concomitantly, very rapidly after DNA damage, and decrease at the same rate thereafter. In the light of this report, our findings suggest that once ATM is activated at a DSB, it could immediately phosphorylate histone H2AX at the site of the break thereby signaling to the cell that a DSB has occurred. This very early event could then initiate the recruitment of DNA repair or damage-signaling factors to the break mediated by chromatin modification and/or direct interactions of these factors with phospho-H2AX. We are grateful to Dr. William Bonner (National Institutes of Health) for the anti-γ-H2AX antibody used in preliminary studies, Dr. Yosef Shiloh (Tel Aviv University) for anti-ATM antibody, and Dr. Martin Lavin (Queensland Institute of Medical Research) for pMat1 plasmid. We thank Melinda Henrie for excellent technical assistance and Steve Yannone, David Gilley, Janice Pluth, and Bipasha Mukherjee for critically reading the manuscript.

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Meiosis-Specific DNA Double-Strand Breaks Are Catalyzed by Spo11, a Member of a Widely Conserved Protein Family

1997-02-01

Scott Keeney , Craig N. Giroux , Nancy Kleckner

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Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of <i>Escherichia coli</i> by R-Factor DNA

1972-08-01

Stanley N. Cohen , Annie Chang , Leslie Hsu

Transformation of E. coli cells treated with CaCl(2) to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial … Transformation of E. coli cells treated with CaCl(2) to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance, fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R-factor DNA in this system.

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A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

2000-08-01

Tanya T. Paull , Emmy P. Rogakou , Vikky Yamazaki +3 more

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DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation

2003-01-01

Christopher J. Bakkenist , Michael B. Kastan

The double-strand-break repair model for recombination

1983-05-01

Jack W. Szostak , Terry L. Orr‐Weaver , Rodney Rothstein +1 more

Causes and consequences of replication stress

2013-12-24

Michelle K. Zeman , Karlene A. Cimprich

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints

2006-11-01

Jiřina Bártková , Nousin Rezaei , Michalis Liontos +7 more

Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes

2003-06-06

Lee Zou , Stephen J. Elledge

The function of the ATR (ataxia-telangiectasia mutated– and Rad3-related)–ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that … The function of the ATR (ataxia-telangiectasia mutated– and Rad3-related)–ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11 , is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

The limited in vitro lifetime of human diploid cell strains

1965-03-01

Leonard Hayflick

A complementation analysis of the restriction and modification of DNA in Escherichia coli

1969-05-01

Herbert W. Boyer , Daisy Roulland-Dussoix

DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis

2005-04-01

Jiřina Bártková , Zuzana Hořejšı́ , Karen Koed +7 more

Positional Cloning of the Werner's Syndrome Gene

1996-04-12

Chang-En Yu , Junko Oshima , Ying‐Hui Fu +7 more

Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The … Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN ) was identified by positional cloning. The predicted protein is 1432 amino acids in length and shows significant similarity to DNA helicases. Four mutations in WS patients were identified. Two of the mutations are splice-junction mutations, with the predicted result being the exclusion of exons from the final messenger RNA. One of these mutations, which results in a frameshift and a predicted truncated protein, was found in the homozygous state in 60 percent of Japanese WS patients examined. The other two mutations are nonsense mutations. The identification of a mutated putative helicase as the gene product of the WS gene suggests that defective DNA metabolism is involved in the complex process of aging in WS patients.

Cancer Susceptibility and the Functions of BRCA1 and BRCA2

2002-01-01

Ashok R. Venkitaraman

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PCNA, the Maestro of the Replication Fork

2007-05-01

George‐Lucian Moldovan , Boris Pfander , Stefan Jentsch

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The DNA Damage Response: Making It Safe to Play with Knives

2010-10-01

Alberto Ciccia , Stephen J. Elledge

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A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.

1989-03-01

M. Litt , J.A. Luty

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions

2005-04-01

Vassilis G. Gorgoulis , Leandros-Vassilios Vassiliou , Panagiotis Karakaidos +7 more

Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo

1999-09-06

Emmy P. Rogakou , Chye Boon , Christophe E. Redon +1 more

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a … The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

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An Oncogene-Induced DNA Damage Model for Cancer Development

2008-03-06

Thanos D. Halazonetis , Vassilis G. Gorgoulis , Jiří Bártek

Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be … Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.

Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG

1991-01-01

Shinya Shibutani , Masaru Takeshita , Arthur P. Grollman

Genome maintenance mechanisms for preventing cancer

2001-05-01

Jan H.J. Hoeijmakers

Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints

2004-06-01

Aziz Sancar , Laura A. Lindsey‐Boltz , Keziban Ünsal-Kaçmaz +1 more

▪ Abstract DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces … ▪ Abstract DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the 9-1-1 complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-S checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed.

Precise Measurement of the G+C Content of Deoxyribonucleic Acid by High-Performance Liquid Chromatography

1989-04-01

M. Mesbah , USHA PREMACHANDRAN , William B. Whitman

High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides … High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by PI nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G+C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C content. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.

DNA Replication in Eukaryotic Cells

2002-06-01

Stephen P. Bell , Anindya Dutta

▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered … ▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage

2007-05-25

Shuhei Matsuoka , Bryan A. Ballif , Agata Smogorzewska +7 more

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion … Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

ATM and related protein kinases: safeguarding genome integrity

2003-02-28

Yosef Shiloh

A simple salting out procedure for extracting DNA from human nucleated cells

1988-01-01

S.A. Miller , Dale D. Dykes , Herbert F. Polesky

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RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO

2002-09-01

Carsten Hoege , Boris Pfander , George‐Lucian Moldovan +2 more

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication

2006-11-01

Raffaella Di Micco , Marzia Fumagalli , Angelo Cicalese +7 more

Identification of a specific telomere terminal transferase activity in tetrahymena extracts

1985-12-01

Carol W. Greider , Elizabeth H. Blackburn

Cell cycle checkpoint signaling through the ATM and ATR kinases

2001-09-01

Robert T. Abraham

The genomes of eukaryotic cells are under continuous assault by environmental agents (e.g., UV light and reactive chemicals) as well as the byproducts of normal intracellular metabolism (e.g., reactive oxygen … The genomes of eukaryotic cells are under continuous assault by environmental agents (e.g., UV light and reactive chemicals) as well as the byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates and inaccurately replicated DNA).Whatever the origin, genetic damage threatens cell survival, and, in metazoans, leads to organ failure, immunodeficiency, cancer, and other pathologic sequelae.

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Instability and decay of the primary structure of DNA

1993-04-01

Tomas Lindahl

Oxidative DNA damage: mechanisms, mutation, and disease

2003-07-01

Marcus S. Cooke , Mark D. Evans , Miral Dizdaroğlu +1 more

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a … Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8-oxo-2'deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.

Molecular mechanisms of cisplatin resistance

2011-09-05

Lorenzo Galluzzi , Laura Senovilla , Ilio Vitale +5 more

Multiple Pathways of Recombination Induced by Double-Strand Breaks in <i>Saccharomyces cerevisiae</i>

1999-06-01

Frédéric Pâques , James E. Haber

SUMMARY The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination … SUMMARY The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.

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The DNA-damage response in human biology and disease

2009-10-20

Stephen P. Jackson , Jiří Bártek

Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction

1986-01-01

Kary B. Mullis , Fred Faloona , S. Scharf +3 more

The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial … The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods.

A Single Ataxia Telangiectasia Gene with a Product Similar to PI-3 Kinase

1995-06-23

Kinneret Savitsky , Anat Bar‐Shira , Shlomit Gilad +7 more

A gene, ATM , that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, … A gene, ATM , that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM , which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3′ kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway

2010-06-07

Michael R. Lieber

Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes … Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes of double-strand breaks (DSBs) result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, DNA polymerases, and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during variable (diversity) joining [V(D)J] recombination and class switch recombination (CSR). Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation (IR), but also severely immunodeficient.

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miR-29a-3p and TGF-β Axis in Fanconi anemia: mechanisms driving metabolic dysfunction and genome stability

2025-06-25

Nadia Bertola , Stefano Regis , Vanessa Cossu +7 more | Cellular and Molecular Life Sciences

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. The FA cellular phenotype is marked by a defective DNA double-strand break repair. Alongside … Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. The FA cellular phenotype is marked by a defective DNA double-strand break repair. Alongside this defect, FA cells exhibit mitochondrial dysfunction and redox unbalance. In addition, FA cells display an altered microRNA profile, including miR-29a-3p, which plays a crucial role in hematopoiesis by supporting the self-renewal, lineage commitment, and differentiation of hematopoietic stem cells (HSCs). In this study, we demonstrate that miR-29a-3p is downregulated in lymphoblasts and fibroblasts mutated for the FANC-A gene, leading to hyperactivation of PI3K/AKT pathway due to the overexpression of its target genes, FOXO3, SGK1, and IGF1, and resulting in altered mitochondrial metabolism and insufficient antioxidant response. In addition, miR-29a-3p downregulation appears associated with hyperactivation of the TGF-β signal. By contrast, FA cells transfected with miR-29a-3p show an improvement in mitochondrial metabolism, oxidative stress response, and DNA damage accumulation, by inhibiting the PI3K/AKT pathway and modulating the TGF-β pathway through a feedback mechanism. In conclusion, our results highlight the central role of miR-29a-3p in FA cells, suggesting that it is a promising molecular target to address several mechanisms based on FA pathogenesis. Graphical abstract

A novel membrane stress response that blocks chromosomal replication by targeting the DnaA initiator via the ClpP protease

2025-06-24

Alabi Gbolahan , Tong Li , Rahul Saxena +5 more | Journal of Bacteriology

In Escherichia coli, membrane stress due to interrupted lipoprotein (Lpp) maturation impairs DNA replication and arrests cell growth. How Lpp maturation and DNA replication are connected remains unclear. We demonstrate … In Escherichia coli, membrane stress due to interrupted lipoprotein (Lpp) maturation impairs DNA replication and arrests cell growth. How Lpp maturation and DNA replication are connected remains unclear. We demonstrate that upon membrane stress, the Rcs stress-response pathway is activated, and the replication initiator DnaA is lost, which explains the replication block. However, Lon protease, a key regulator of the Rcs pathway, is not required for the DnaA loss. We further ruled out the involvement of (p)ppGpp, one of the major mediators of stress responses in bacteria. On the other hand, upon deletion of the ClpP protease gene, DnaA was stable, replication was not inhibited, and there was no cell-growth arrest. In wild-type cells, overexpression of DnaA was lethal even without the membrane stress apparently from hyperinitiation. In ∆crp cells, hyperinitiation was restrained, and overexpression of DnaA was able to overcome the growth arrest. ∆fis cells, which were earlier found resistant to the membrane stress, showed DnaA stability and normal replication upon stress-inducing treatments. We conclude that DnaA loss suffices to explain the growth arrest upon the membrane stress. The stress-response pathway described here appears novel because of its independence from Lon and (p)ppGpp, which have been implicated in other stress responses that block DNA replication. The observation that DNA replication stress can block cell division in E. coli (SOS response) introduced the concept of checkpoint control in the cell cycle. Here, we describe a novel checkpoint control that functions in the opposite direction: membrane stress causing replication block. We show how the accumulation of precursor lipoprotein (pLpp) could block replication. The pLpp accumulation causes a response culminating in activating the ClpP protease that blocks replication by targeting the initiator DnaA. DnaA being vital and highly conserved, a detailed understanding of the response pathway is likely to open new avenues to treat bacterial infection.

Mechanostress-induced DNA damage in migrating neurons during brain development

2025-06-23

Mineko Kengaku , Zhejing Zhang , Andrés Canela +7 more | Research Square (Research Square)

<title>Abstract</title> Migratory cells tend to have soft nuclei that deform and penetrate narrow spaces. Extensive nuclear deformation during migration can cause nuclear envelope rupture and DNA damage in cancer cells, … <title>Abstract</title> Migratory cells tend to have soft nuclei that deform and penetrate narrow spaces. Extensive nuclear deformation during migration can cause nuclear envelope rupture and DNA damage in cancer cells, which may contribute to the malignant transformation during tumor progression. However, the significance of DNA damage in physiological migration is less well understood. Here, we demonstrate that the migration of neurons in developing cerebral and cerebellar cortices is accompanied by massive DNA double-strand breaks (DSBs) due to mechanostress during passage through narrow interstitial spaces. Confined migration enhances the binding and cleavage of the genome by topoisomerase IIbeta expressed in neuronal nucleus, independently of the nuclear envelope rupture. Genome sequencing revealed that DSBs tend to occur outside of protein-coding regions and transcription regulatory regions. During normal development, DSBs are rapidly repaired by the non-homologous end joining pathway. The deletion of ligase IV at the onset of neuronal migration leads to persistent DSB accumulation in cerebellar neurons with moderate transcriptional changes in genes related to synaptic function, neuronal development, and stress and immune responses. The mutant mouse develops mild motor deficits in later life, suggesting that the DNA damage generated during normal brain development poses a potential disease risk if left unrepaired.

Evidence for a transgenerational mutational signature from ionizing radiation exposure in humans

2025-06-23

Fabian Brand , Hannah Klinkhammer , Alexej Knaus +7 more | Scientific Reports

The existence of transgenerational effects of radiation exposure on the human germline remains controversial. Evidence for transgenerational biomarkers are of particular interest for populations, who have been exposed to higher … The existence of transgenerational effects of radiation exposure on the human germline remains controversial. Evidence for transgenerational biomarkers are of particular interest for populations, who have been exposed to higher than average levels of ionizing radiation (IR). This study investigated signatures of parental exposure to IR in offspring of former German radar operators and Chernobyl cleanup workers, focusing on clustered de novo mutations (cDNMs), defined as multiple de novo mutations (DNMs) within 20 bp. We recruited 110 offspring of former German radar operators, who were likely to have been exposed to IR (Radar cohort, exposure = 0-353 mGy), and reanalyzed sequencing data of 130 offspring of Chernobyl cleanup workers (CRU, exposure = 0-4080 mGy) from Yeager, et al. In addition, we analyzed whole genome trio data of 1275 offspring from unexposed families (Inova cohort). We observed on average 2.65 cDNMs (0.61 adjusted for the positive predictive value (PPV)) per offspring in the CRU cohort, 1.48 (0.34 PPV) in the Radar cohort and 0.88 (0.20 PPV) in the Inova cohort. Although under the condition that the proportion of true mutations is low in this analysis, this represented a significant increase ([Formula: see text]) of cDNMs counts, that scaled with paternal exposure to IR ([Formula: see text]). Our findings corroborate that cDNMs are a potential transgenerational biomarker of paternal IR exposure.

Targeting DNA Damage Response-Mediated Resistance in Non-Small Cell Lung Cancer: From Mechanistic Insights to Drug Development

2025-06-23

Xue Gong , Yongzhao Zhou , Yi Guo Deng | Current Oncology

Non-small cell lung cancer (NSCLC) remains a major contributor to cancer-related deaths worldwide, with therapeutic resistance presenting a critical clinical hurdle. The DNA damage response (DDR) constitutes a sophisticated cellular … Non-small cell lung cancer (NSCLC) remains a major contributor to cancer-related deaths worldwide, with therapeutic resistance presenting a critical clinical hurdle. The DNA damage response (DDR) constitutes a sophisticated cellular framework that detects, signals, and repairs genetic lesions to preserve genomic stability. While the DDR plays a crucial role in determining the efficacy of radiotherapy and chemotherapy, current research primarily focuses on direct DDR inhibitors, often overlooking the broader regulatory networks that modulate DDR activity. This review aims to comprehensively analyze the upstream and downstream pathways governing DDR in NSCLC, highlighting key molecular regulators, signaling interactions, and potential feedback mechanisms contributing to therapy resistance. By identifying novel regulatory targets and clinically relevant biomarkers, we propose innovative therapeutic strategies to enhance treatment efficacy. Our approach seeks to bridge the gap between DDR dysregulation and precision oncology, offering new perspectives on overcoming resistance and improving patient outcomes in NSCLC.

Vimentin intermediate filaments orchestrate DNA nonhomologous end joining repair and lipolysis after DNA damage

2025-06-23

Feifei Wang , Mengtao Rong , Liang Zhang +7 more | Oncogene

The Yin and Yang of replicative aging and rejuvenation

2025-06-23

Wei‐Han Lin , Aliaksandr Damenikan , Yves Barral | Current Opinion in Genetics & Development

Budding yeast undergoes replicative aging through asymmetric cell divisions. Yeast mother cells progressively age as they generate successive daughter cells, until they ultimately die. However, their daughters are born rejuvenated, … Budding yeast undergoes replicative aging through asymmetric cell divisions. Yeast mother cells progressively age as they generate successive daughter cells, until they ultimately die. However, their daughters are born rejuvenated, that is, most of them recover a full lifespan potential, with little impact of their mothers' age at their birth. In this review, we will discuss recent findings regarding the mechanisms of replicative aging and rejuvenation. Based on these insights, we will also discuss which evolutionary forces may have presided over the emergence of aging in yeast. We suggest that aging and rejuvenation represent two adaptive strategies that each bring their own benefits.

Correction: Replication of BRIT1/MCPH1 G2/M Checkpoint Function Confirms Original Conclusions

2025-06-22

Shiaw‐Yih Lin , Hui Dai , Stephen J. Elledge | bioRxiv (Cold Spring Harbor Laboratory)

In our 2005 PNAS publication, we reported that BRIT1/MCPH1 is required for proper activation of the G2/M checkpoint in response to DNA damage. A recent analysis using image comparison software, … In our 2005 PNAS publication, we reported that BRIT1/MCPH1 is required for proper activation of the G2/M checkpoint in response to DNA damage. A recent analysis using image comparison software, Proofig, identified a duplicated panel in Figure 2A, stemming from a figure assembly error in which data from a prior experiment involving a different gene (Claspin) were inadvertently reused. As the original data are no longer accessible due to the age of the experiment, we have independently repeated the G2/M checkpoint analysis. The new results fully confirm the original conclusion: BRIT1 depletion compromises G2/M arrest following ionizing radiation. This replication supports the integrity of our original findings and is provided in support of a formal correction requested by PNAS.

Development of translational read-through-inducing drugs as novel therapeutic options for patients with Fanconi anemia

2025-06-21

Anca Manuela Hristodor , Enrico Cappelli , Elena Baldisseri +7 more | Cell Death Discovery

Abstract Fanconi anemia (FA) is caused by mutations affecting FANC genes involved in DNA repair, with nearly 20% of FA patients harboring nonsense mutations. Ataluren (PTC124) is a translational read-through-inducing … Abstract Fanconi anemia (FA) is caused by mutations affecting FANC genes involved in DNA repair, with nearly 20% of FA patients harboring nonsense mutations. Ataluren (PTC124) is a translational read-through-inducing drug (TRID) already approved in Europe that has a well-established safety profile even in pediatric patients. Amlexanox, an anti-inflammatory drug, also promotes read-through of premature stop codons caused by nonsense mutations. We compared ataluren and amlexanox in rescuing FANCA, FANCC and FANCF protein synthesis in lymphoblastoid cell lines and fibroblasts obtained from FA patients with nonsense mutations. While ataluren restored all FANC protein levels, amlexanox was partially effective only on FANCA. Notably, the rescue of FANC proteins resulted in a significant downregulation of p53. Moreover, unlike amlexanox, ataluren remarkably improved cell viability and reduced chromosomal aberrations upon exposure to genotoxic compounds. Amlexanox primarily reduced the signal transducer and activator of transcription 2 (STAT2) phosphorylation. Furthermore, FANCA -mutated fibroblasts exhibited a higher frequency of micronuclei formation as well as lower lamin B1 expression compared to their gene-edited counterpart re-expressing wild-type FANCA . Interestingly, ataluren significantly limited the generation of micronuclei in nonsense-mutated primary FANCC fibroblasts, restoring lamin B1 expression. This study represents a milestone of drug development for FA as it paves the way for clinical development of TRIDs, indicating ataluren as a promising approach to address the genetic instability and reduce the risk of malignant transformation in FA cells. Moreover, these results highlight the importance of a reliable experimental pipeline to assess whether minimal protein rescue via translational read-through can yield meaningful phenotypic rescue.

A four-in-one replicase integrating key enzymatic activities for DNA replication.

2025-06-20

Yuxin Zhang , Xueling Lu , Bin Zhu +1 more | PubMed

DNA replication is a fundamental process in all living organisms. As the most diverse and abundant biological entities on Earth, bacteriophages may utilize unconventional methods for genome replication. In this … DNA replication is a fundamental process in all living organisms. As the most diverse and abundant biological entities on Earth, bacteriophages may utilize unconventional methods for genome replication. In this study, we identified a novel DNA replicase, GP55, from lactococcal phage 1706. GP55 comprises a helicase domain, a distinctive archaeo-eukaryotic primase domain, and a family B DNA polymerase domain, collectively exhibiting helicase, primase, and DNA polymerase activities, along with intrinsic 3'-5' exonuclease activity. Notably, the helicase activity of GP55 is UTP/dTTP-dependent rather than ATP-dependent and facilitates strand displacement during DNA synthesis. GP55 exhibits a unique primase activity, recognizing specific but less stringent DNA sequences and preferring GTP for the initiation of RNA primer synthesis. Additionally, a newly identified α-helix domain, composed of two pairs of parallel α-helices, was found to be essential for its primase activity. The multiple activities enable GP55 to efficiently synthesize DNA de novo in the presence of dNTPs and NTPs. This study reveals a concise strategy employed by bacteriophages for genome replication using multifunctional replicases.

Quantification of liver fat fraction using T1-weighted mDixon MRI in young patients with ataxia telangiectasia undergoing whole-body MRI: an exploratory study

2025-06-20

Soma Kumasaka , Rafał Panek , Renata Neves +5 more | Orphanet Journal of Rare Diseases

Abstract Background Ataxia–telangiectasia (A–T) is an inherited multiorgan disorder with onset in childhood. Liver involvement, with steatosis and subsequent fibrosis, is increasingly recognized in children and young people with A–T. … Abstract Background Ataxia–telangiectasia (A–T) is an inherited multiorgan disorder with onset in childhood. Liver involvement, with steatosis and subsequent fibrosis, is increasingly recognized in children and young people with A–T. Purpose To evaluate feasibility of T1-weighted two-point mDixon MRI for identification of liver steatosis in children with A–T and conduct exploratory analysis of relationships between MRI-quantified liver fat fraction and clinical and laboratory measures. Study type Post hoc analysis of prospectively collected research data. Population 16 participants (8 female) with A–T aged 4.8–16.6 years. Field strength/sequence 3.0-T, two-point T1-weighted mDixon. Assessment Participants underwent whole-body MRI including T1-weighted mDixon. Water/fat signal percentage images were generated. Hepatic T1 fat fraction (T1-FF) was calculated from regions-of-interest placed in the right anterior, right posterior and left hepatic lobes. T1-FF > 5.56% was used as the diagnostic criterion for hepatic steatosis. Statistical tests Group comparisons of variables between participants with and without previous diagnosis of liver steatosis were made using independent sample Mann–Whitney U. Associations between T1-FF and age, neurological severity and of liver function tests were tested with Spearman correlation. Statistical significance was pre-specified as p < 0.05. Results Analyzable T1-weighted mDixon data was available for 11 participants. Five MRI datasets were discarded due to motion artefact (n = 3) or incorrect archiving of the original water image (n = 2). Median liver T1-FF was 11.3% (4.7–49.7%), and 10/11 (91%) of participants had evidence of steatosis. Participants with previous diagnosis of steatosis had higher T1-FF than those without (median 32.7% [9.7–49.7%], versus 10.3% [4.7–11.3%], p = 0.030). T1-FF correlated most strongly with alanine transaminase (r = 0.76, p = 0.007) and γ-glutamyltransferase (r = 0.76, p = 0.006). Conclusion T1-weighted mDixon MRI is feasible for detecting steatosis in children with A–T, although motion artefacts reduced data completeness. MRI-quantified liver T1-FF correlates with markers of liver health. We found higher prevalence of liver steatosis using T1-weighted mDIXON than previously reported in pediatric A–T cohorts.

Senataxin and DNA-PKcs redundantly promote non-homologous end joining repair of DNA double strand breaks during V(D)J recombination

2025-06-20

Bo-Ruei Chen , Thu Pham , Lance D. Reynolds +7 more | Science Advances

Nonhomologous end joining (NHEJ) is required for repairing DNA double strand breaks (DSBs) generated by the RAG endonuclease during lymphocyte antigen receptor gene assembly by V(D)J recombination. The ataxia telangiectasia–mutated … Nonhomologous end joining (NHEJ) is required for repairing DNA double strand breaks (DSBs) generated by the RAG endonuclease during lymphocyte antigen receptor gene assembly by V(D)J recombination. The ataxia telangiectasia–mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) kinases regulate functionally redundant pathways required for NHEJ. Here, we report that loss of the senataxin helicase leads to a strong defect in RAG DSB repair upon inactivation of DNA-PKcs. The NHEJ function of senataxin is redundant with the RECQL5 helicase and the HLTF translocase and is epistatic with ATM. Co-inactivation of ATM, RECQL5, and HLTF results in an NHEJ defect similar to that from the combined deficiency of DNA-PKcs and senataxin or losing senataxin, RECQL5, and HLTF. These data suggest that ATM and DNA-PKcs regulate the functions of senataxin and RECQL5/HLTF, respectively, to provide redundant support for NHEJ.

Polymerase theta expression is correlated with proliferative capacity but not with DNA repair deficiency status in solid tumors

2025-06-20

Zsófia Sztupinszki , Regina Nóra Fiam , Orsolya Pipek +5 more | npj Precision Oncology

Polymerase theta (POLθ) inhibitors were developed to overcome resistance to PARP inhibitor treatment in homologous recombination (HR) deficient cancer. Biomarkers identifying PARP inhibitor-resistant cancer cases that specifically rely on POLθ … Polymerase theta (POLθ) inhibitors were developed to overcome resistance to PARP inhibitor treatment in homologous recombination (HR) deficient cancer. Biomarkers identifying PARP inhibitor-resistant cancer cases that specifically rely on POLθ activity for cancer cell survival will help to identify the sensitive patient population. From the TCGA RNAseq data, we determined POLθ expression levels, and from whole-exome and whole-genome sequencing data, we determined the number of POLθ-associated mutational signatures in solid tumors with various HR deficiency status. We found that POLθ expression levels did not differ significantly between HR-proficient and HR-deficient cancers. However, POLθ expression correlated strongly with proliferation-associated gene expression signatures and was predominantly observed in the S and G2/M phases of the cell cycle. POLθ-associated mutational signatures are correlated with POLθ expression levels only in BRCA2-deficient cancers. POLθ expression level and POLθ-associated mutational signatures may be indicative of POLθ inhibitor sensitivity in BRCA2-deficient tumors, but are unlikely to be informative in other cancers.

Correction to 'Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases'.

2025-06-20

| PubMed

DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

2025-06-20

Sazzad Khan , Himanshi Singh , Jianfeng Xiao +1 more | Biomolecules

Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by the degeneration of dopaminergic neurons in the substantia nigra, leading to decreased dopamine levels in the striatum and causing a … Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by the degeneration of dopaminergic neurons in the substantia nigra, leading to decreased dopamine levels in the striatum and causing a range of motor and non-motor impairments. Although the molecular mechanisms driving PD progression remain incompletely understood, emerging evidence suggests that the buildup of nuclear DNA damage, especially DNA double-strand breaks (DDSBs), plays a key role in contributing neurodegeneration, promoting senescence and neuroinflammation. Despite the pathogenic role for DDSB in neurodegenerative disease, targeting DNA repair mechanisms in PD is largely unexplored as a therapeutic approach. Ataxia telangiectasia mutated (ATM), a key kinase in the DNA damage response (DDR), plays a crucial role in neurodegeneration. In this study, we evaluated the therapeutic potential of AZD1390, a highly selective and brain-penetrant ATM inhibitor, in reducing neuroinflammation and improving behavioral outcomes in a mouse model of α-synucleinopathy. Four-month-old C57BL/6J mice were unilaterally injected with either an empty AAV1/2 vector (control) or AAV1/2 expressing human A53T α-synuclein to the substantia nigra, followed by daily AZD1390 treatment for six weeks. In AZD1390-treated α-synuclein mice, we observed a significant reduction in the protein level of γ-H2AX, a DDSB marker, along with downregulation of senescence-associated markers, such as p53, Cdkn1a, and NF-κB, suggesting improved genomic integrity and attenuation of cellular senescence, indicating enhanced genomic stability and reduced cellular aging. AZD1390 also significantly dampened neuroinflammatory responses, evidenced by decreased expression of key pro-inflammatory cytokines and chemokines. Interestingly, mice treated with AZD1390 showed significant improvements in behavioral asymmetry and motor deficits, indicating functional recovery. Overall, these results suggest that targeting the DDR via ATM inhibition reduces genotoxic stress, suppresses neuroinflammation, and improves behavioral outcomes in a mouse model of α-synucleinopathy. These findings underscore the therapeutic potential of DDR modulation in PD and related synucleinopathy.

DNA Damage and Replication Stress Checkpoints

2025-06-20

Luke A. Yates , Xiaodong Zhang , Peter Burgers | Annual Review of Biochemistry

DNA damage checkpoints are key regulatory signaling cascades that arrest cell cycle progression upon DNA damage or upon DNA replication stalling and allow time for repair or correction. Failure to … DNA damage checkpoints are key regulatory signaling cascades that arrest cell cycle progression upon DNA damage or upon DNA replication stalling and allow time for repair or correction. Failure to elicit these checkpoints can lead to genomic instability that can result in cell death or mutations, ultimately leading to diseases such as cancer. Components of the DNA damage checkpoint are attractive targets for precision medicine to treat cancers. Over the last several years, cutting-edge structural techniques have provided molecular insights into the highly coordinated checkpoint signaling that occurs in response to DNA damage or other obstacles to replication progression. This review summarizes our current mechanistic understanding of the DNA damage checkpoint in eukaryotes, with an emphasis on the sensor kinases ATM (Tel1) and ATR (Mec1), highlighting structure–function and cellular studies.

DNAJB6: A guardian against neurodegeneration

2025-06-19

Jónvá Hentze , Anna C. Gelman , Tomasz Brudek +1 more | Neural Regeneration Research

Abstract Amyloid protein aggregation plays a major role in multiple neurodegenerative diseases and is likely the primary driving force for the progression of most of these diseases. Multiple recent studies … Abstract Amyloid protein aggregation plays a major role in multiple neurodegenerative diseases and is likely the primary driving force for the progression of most of these diseases. Multiple recent studies have highlighted that the DNAJ homolog subfamily B member 6 (DNAJB6) chaperone is particularly interesting, when it comes to preventing amyloidogenic proteins from aggregating. It has been shown that DNAJB6 can prevent the aggregation of polyglutamine-expanded proteins in models of Huntington’s disease. Likewise, it can suppress aggregation of α-synuclein in models of Parkinson’s disease and other synucleinopathies. Finally, it has been shown that DNAJB6 can block aggregation of multiple additional amyloid proteins involved in Alzheimer’s disease and other tauopathies as well. We believe there is yet much to learn about the protective role of DNAJB6 in the brain, but this focused review summarizes, what we know so far of this chaperone. It describes the biological role of DNAJB6 in the brain and its interaction with Hsp70, with particular emphasis on the studies that show its ability to prevent amyloid protein aggregation in vitro and in vivo . Moreover, recent work on dysregulation of the expression of DNAJB6 in brain clinical tissue is discussed. Finally, we discuss potential therapeutic perspectives as we believe this protein is a promising druggable target.

Insights Into Cockayne Syndrome Type B: What Underlies Its Pathogenesis?

2025-06-19

Ricardo Afonso‐Reis , Carolina Madeira , David V. C. Brito +1 more | Aging Cell

ABSTRACT Cockayne Syndrome (CS) is an autosomal recessive disorder arising from mutations in either of two disease‐associated genes, ERCC6 or ERCC8 . CS patients exhibit cutaneous photosensitivity, neuropathological abnormalities, severe … ABSTRACT Cockayne Syndrome (CS) is an autosomal recessive disorder arising from mutations in either of two disease‐associated genes, ERCC6 or ERCC8 . CS patients exhibit cutaneous photosensitivity, neuropathological abnormalities, severe growth retardation, a distinctive facial appearance with pronounced sunken eyes, musculoskeletal anomalies, sensory impairment, and dental decay. Approximately 70% of all CS cases carry ERCC6 mutations; therefore, this review will focus solely on Cockayne Syndrome complementation group B (CS‐B). CS‐B exhibits several hallmarks of aging, including genomic instability, epigenetic modifications, loss of proteostasis, and mitochondrial failure. CS‐B is proposed to result from the accumulation of DNA damage and the resulting transcription impairment. However, the main pathophysiological mechanisms underlying the severe cellular impairments observed in CS‐B remain unclear. Here, we review the current literature to elucidate ERCC6‐related mechanisms, highlighting the key and emerging pathological mechanisms underlying CS‐B, as well as their putative interactions. Considering the mechanisms that heavily rely on ERCC6, we propose that CS‐B pathogenesis arises from a combination of DNA damage accumulation, transcriptional dysregulation, and mitochondrial dysfunction. Furthermore, we argue that these molecular features influence each other, rather than acting as isolated mechanisms. This suggests that the crosstalk between mechanisms is a key factor for CS‐B pathogenesis. Although efforts have been made to unveil CS‐B pathogenesis, research is still lacking, hindering progress in understanding this deadly disease. Future work will prove crucial to determine the main contributor to CS‐B pathogenesis and identify new interactions between CS‐B‐affected mechanisms.

DNA polymerase iota alleviates replication stress-induced genome instability during mitosis

2025-06-19

Lei Li , Can Yi , Silu Cheng +7 more | Research Square (Research Square)

<title>Abstract</title> DNA polymerase iota (Pol ι) is a Y-family lesion bypass polymerases, characterized by its remarkably low fidelity. While Pol ι is ubiquitously expressed in nearly all cell types, its … <title>Abstract</title> DNA polymerase iota (Pol ι) is a Y-family lesion bypass polymerases, characterized by its remarkably low fidelity. While Pol ι is ubiquitously expressed in nearly all cell types, its function remains largely undefined. Previous studies in cellular and mouse models deficient in Pol ι have revealed no discernible phenotype. In this report, we show that Pol ι plays an important role in mitotic DNA synthesis. Cells lacking Pol ι exhibit heightened vulnerability to replication stress, increased formation of micronuclei, and accumulation of chromosomal breaks. The protein level of Pol ι is tightly regulated and restricted to G2/M phase cells via CRL7/FBXW11-mediated proteasomal degradation. The absence of Pol ι during S phase ensures that its error-prone DNA synthesis activity is excluded from interfering with normal DNA replication. These findings suggest that Pol ι function is critical in resolving DNA damage-induced replication stress prior to the completion of mitosis.

Unlocking the chemistry facilitated by enzymes that process nucleic acids using quantum mechanical and combined quantum mechanics–molecular mechanics techniques

2025-06-19

Dylan J. Nikkel , Stacey D. Wetmore | Pure and Applied Chemistry

Abstract The diverse cellular functions of nucleic acids are made possible by enzymes that catalyze cleavage of glycosidic (nucleobase–sugar) and phosphodiester bonds. Despite advancements in experimental biochemical methods, critical information … Abstract The diverse cellular functions of nucleic acids are made possible by enzymes that catalyze cleavage of glycosidic (nucleobase–sugar) and phosphodiester bonds. Despite advancements in experimental biochemical methods, critical information about such enzyme-catalyzed reactions is difficult to obtain from traditional experiments. However, computational quantum mechanical (QM) methods can provide atomic level details of catalytic pathways that are complementary to experimental data. This perspective highlights various QM techniques used to advance our understanding of enzymes that process nucleic acids. First, select DNA glycosylases are discussed to showcase how QM calculations on nucleoside/tide and small molecule complexes uncover roles of active site interactions and the preferred order of reaction steps along DNA repair pathways. Furthermore, the ability of calculations on nucleic acid–enzyme complexes that combine QM methods with molecular mechanics (MM) force fields to challenge traditional views of enzyme function and lead to consensus for mechanistic pathways is illustrated. Subsequently, QM-based studies of select nucleases are discussed to highlight how this methodology can discern the various strategies enzymes use to cleave nucleic acid backbones. Overall, this contribution underscores the value in combining QM-based computational work with experimental studies to uncover enzyme-facilitated nucleic acid chemistry to be harnessed in future medicinal, biotechnological and materials applications.

Protein–Protein Interactions in Base Excision Repair

2025-06-18

Govardhan Rathnaiah , Joann B. Sweasy | Biomolecules

The Base Excision Repair (BER) pathway involves a highly coordinated series of protein-protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, … The Base Excision Repair (BER) pathway involves a highly coordinated series of protein-protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase-phosphatase (PNKP), DNA polymerase b (Pol β), ligase IIIα (LigIIIα), poly (ADP-ribose) polymerases PARP1 and PARP2, and X-ray repair cross-complementing protein 1 (XRCC1) catalyze BER in a tightly regulated molecular network. These interactions ensure the seamless handoff of DNA intermediates between the core enzymes of the BER pathway. Understanding the details of protein-protein interactions in BER provides valuable insights into the molecular underpinnings of DNA repair processes. In this review, we focus on protein-protein interactions between the components of the single-nucleotide BER (SN-BER) pathway and other proteins that interact with BER components and regulate the coordination of the pathway. We also briefly discuss the interactions of other proteins that interact with the components of SN-BER based on functional evidence.

Deep structure–function analysis of the endonuclease Mus81 with dominant mutational scanning

2025-06-18

Anthony Oppedisano , Melanie L. Bailey , Arun Kumar +3 more | Proceedings of the National Academy of Sciences

Protein structure–function relationships are critical for understanding molecular mechanisms and the impacts of genetic variation. Mutational scanning approaches can deliver scalable analysis, usually through the study of loss-of-function variants. Rarer … Protein structure–function relationships are critical for understanding molecular mechanisms and the impacts of genetic variation. Mutational scanning approaches can deliver scalable analysis, usually through the study of loss-of-function variants. Rarer dominant negative and gain-of-function variants can be more information rich, as they retain a stable proteoform and can be used to dissect molecular function while retaining biological context. Dominant variant proteoforms can still engage substrates and interact with binding partners. Here, we probe the structure–function relationships of the Mus81 endonuclease by ectopic expression of deep mutational scanning libraries to find amino acid variants that confer dominant sensitivity to genotoxic stress and dominant synthetic lethality. Screening more than 2,200 MUS81 variants at 100 positions identified 13 amino acids that can be altered to elicit a dominant phenotype. The dominant phenotype of these variants required the presence of the obligate Mus81 binding protein, Mms4. The dominant variants affect amino acids in a contiguous surface on Mus81 and fall into two distinct classes: residues that bind the catalytic magnesium atoms and residues that form the hydrophobic wedge. Most of the variant amino acids were conserved across species and cognate variants expressed in human cell lines resulted in dominant sensitivity to replication stress and synthetic growth defects in cells lacking BLM helicase. The dominant variants in both yeast and human MUS81 resulted in phenotypes distinct from a MUS81 knockout. These data demonstrate the utility of dominant genetics using ectopic expression of amino acid site saturation variant libraries to link function to protein structure providing insight into molecular mechanisms.

Xeroderma Pigmentosum in Identical Twins: Synchronous Presentation of Distinct Malignancies

2025-06-18

Swapnil Kaushal , Jaydeep N Pol , Dipti Patil +4 more | Indian Journal of Otolaryngology and Head & Neck Surgery

Proteasome-dependent Orc6 removal from chromatin upon S-phase entry safeguards against MCM reloading and tetraploidy

2025-06-18

Yoko Hayashi‐Takanaka , Ichiro Hiratani , Tokuko Haraguchi +1 more | Journal of Cell Science

DNA replication is tightly regulated to occur only once per cell cycle, as untimely re-initiation can lead to aneuploidy, which is associated with early senescence and cancer. The pre-replication complex … DNA replication is tightly regulated to occur only once per cell cycle, as untimely re-initiation can lead to aneuploidy, which is associated with early senescence and cancer. The pre-replication complex (comprising Orc1-6, Cdc6, Cdt1, and MCM) is essential for the initiation of DNA replication, but the dynamics and function of Orc6 during the cell cycle remain elusive. Here, we demonstrate that Orc6 associates with chromatin during G1-phase and dissociates upon S-phase entry. The dissociation of Orc6 from chromatin is dependent on proteasome activity, and inhibition of the proteasome leads to the accumulation of chromatin-bound Orc6, which promotes abnormal MCM loading after S-phase entry without undergoing mitosis in human immortalized hTERT-RPE1 cells. Following release from proteasome inhibition, cells with elevated levels of chromatin-bound Orc6 and MCM proceed to the next replication phase as tetraploid cells. Our findings suggest that the proteasome-dependent dissociation of Orc6 after DNA replication is critical for preventing inappropriate MCM reloading and tetraploid formation.

UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

2025-06-17

Zijuan Wang , Benjamin M. Foster , Isabelle Cristine da Costa +7 more | bioRxiv (Cold Spring Harbor Laboratory)

SUMMARY DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify … SUMMARY DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify UFM1 as a previously unrecognised regulator of non-homologous end-joining (NHEJ). Using a structure-guided chemical biology strategy, we develop a photo-crosslinkable UFM1 probe and, together with high-resolution NMR, uncover non-canonical UFM1-binding regions in core NHEJ components, including XRCC4. Mechanistically, proximity-dependent proteomics reveals Ku70 as a key UFMylation substrate, establishing a functional axis in which XRCC4 engages UFMylated Ku70 to promote the chromatin assembly of NHEJ factors. Perturbation of UFM1 signalling, via UFSP2 depletion or a hypomorphic UBA5 allele in patient-derived fibroblasts, impairs these processes, linking UFMylation defects to altered regulation of DSB repair. Our findings define a complete UFM1 signalling module in genome maintenance and uncover a molecular connection between hereditary UFMylation disorders and dysregulated DSB repair pathways.

Comprehensive Measurement of Inter-Individual Variation in DNA Repair Capacity in Healthy Individuals

2025-06-16

Ting Zhai , Patrizia Mazzucato , Catherine Ricciardi +5 more | medRxiv (Cold Spring Harbor Laboratory)

Rare genetic DNA repair deficiency syndromes can lead to immunodeficiency, neurological disorders, and cancer. In the general population, inter-individual variation in DNA repair capacity (DRC) influences susceptibility to cancer and … Rare genetic DNA repair deficiency syndromes can lead to immunodeficiency, neurological disorders, and cancer. In the general population, inter-individual variation in DNA repair capacity (DRC) influences susceptibility to cancer and several age-related diseases. Genome wide association studies and functional analyses show that defects in multiple DNA repair pathways jointly increase disease risk, but previous technologies did not permit comprehensive analyses of DNA repair in populations. To overcome these limitations, we used fluorescence multiplex host cell reactivation (FM-HCR) assays that directly quantify DRC across six major DNA repair pathways. We assessed DRC in phytohemagglutinin-stimulated primary lymphocytes from 56 healthy individuals and validated assay reproducibility in 10 individuals with up to five independent blood draws. We furthermore developed generalized analytical pipelines for systematically adjusting for batch effects and both experimental and biological confounders. Our results reveal significant inter-individual variation in DRC for each of 10 reporter assays that measure the efficiency of distinct repair processes. Our data also demonstrate that correlations between the activities of different DNA repair pathways are relatively weak. This finding suggests that each pathway may independently influence susceptibility to the health effects of DNA damage. We furthermore developed a pipeline for analyzing comet repair kinetics and related our new functional data to previously reported comet assay data for the same individuals. Our pioneering analysis underscores the sensitivity of FM-HCR assays for detecting subtle biological differences between individuals and establishes standardized methodologies for population studies. Our findings and open source analytical tools advance precision medicine by enabling comprehensive exploration of genetic, demographic, clinical, and lifestyle factors and supporting targeted interventions to enhance DNA repair and maintain genomic integrity, thereby promoting personalized healthcare and disease prevention.

Ataxin-2 preserves oocyte genome integrity by promoting timely premeiotic DNA replication

2025-06-16

Vernon Monteiro , C. W. Yu , Camilla Roselli +3 more | bioRxiv (Cold Spring Harbor Laboratory)

The faithful reassortment and transmission of chromosomes across generations is fundamental to species survival. While much is known about chromosome pairing and recombination, the upstream regulators controlling entry into the … The faithful reassortment and transmission of chromosomes across generations is fundamental to species survival. While much is known about chromosome pairing and recombination, the upstream regulators controlling entry into the meiotic program remain largely elusive. In many species, including Drosophila and mammals, the decision to enter meiosis occurs prior to premeiotic DNA replication and is governed by post-transcriptional regulation, by yet to be discovered factors. Here, we identify the RNA-binding protein Ataxin-2 as a crucial and previously unrecognized regulator of meiotic entry. We show that Ataxin-2 acts post-transcriptionally to promote the entry into meiosis by downregulating the conserved cell cycle inhibitor Dacapo, the Drosophila ortholog of p21/p27. In the absence of Ataxin-2, germ cells mis-regulate Dacapo leading to delayed premeiotic DNA replication and sterility. Strikingly, when DNA replication is delayed and extends into the next stage of meiosis, synaptonemal complex formation, oocytes incur severe genomic DNA damage, likely caused by collisions between replication forks and the synaptonemal complex. Our findings establish Ataxin-2 as a pivotal factor in regulating premeiotic DNA replication and safeguarding oocyte genome stability, shedding new light on the intricate regulatory mechanisms that ensure successful meiosis and fertility.

Modulation of SUMO1-TOP1 DNA damage repair by TOPORS following ovalbumin-induced oxidative stress in macrophages

2025-06-16

Xulong Cai , Qiaolan Xu , Tongjin Yin +4 more | Toxicology Letters

<scp>RECQ4</scp> restricts non‐interfering crossover formation to fine‐tune meiotic recombination rates in rice

2025-06-15

Yafei Li , Hanli You , Xinjie Cheng +7 more | Plant Biotechnology Journal

Summary Crop breeding fundamentally depends on meiotic crossovers (COs) to reshuffle genetic material and integrate favourable alleles into elite cultivars. Recombination frequency is of paramount importance in this process. Higher … Summary Crop breeding fundamentally depends on meiotic crossovers (COs) to reshuffle genetic material and integrate favourable alleles into elite cultivars. Recombination frequency is of paramount importance in this process. Higher recombination rates enhance the probability of breaking linkage drag and generating novel allelic combinations. Here, using rice ( Oryza sativa ) as a model crop species, we reveal that RECQ4, a conserved suppressor of meiotic CO formation, is indispensable for safeguarding the integrity of meiotic recombination intermediate metabolism. We demonstrate that RECQ4 limits COs in rice by specifically suppressing non‐interfering CO pathways. Genetic redundancy with FANCM underscores their cooperative function in ensuring canonical CO formation, which is essential for accurate homologue segregation and genome stability. Furthermore, the recq4 dmc1 double mutant exhibits persistent chromosome fragmentation, implicating RECQ4 in resolving recombination intermediates through sister chromatid repair. Our findings redefine RECQ4's role in crop meiosis, bridging its CO suppression activity with broader genome surveillance functions.

Oxamate, an LDHA Inhibitor, Inhibits Stemness, Including EMT and High DNA Repair Ability, Induces Senescence, and Exhibits Radiosensitizing Effects in Glioblastoma Cells

2025-06-14

Takuma Hashimoto , Go Ushikubo , Naoya Arao +3 more | International Journal of Molecular Sciences

Enhancement of glycolysis has been reported in tumor cells, and it is believed that this enhancement is important for maintaining the stemness of tumor cells and contributes to malignant phenotypes. … Enhancement of glycolysis has been reported in tumor cells, and it is believed that this enhancement is important for maintaining the stemness of tumor cells and contributes to malignant phenotypes. Here, we investigated the effects of Oxamate, which inhibits glycolysis by blocking the conversion of pyruvate to lactate, on radiosensitivity and its molecular mechanisms in T98G glioblastoma cells. Oxamate significantly enhanced radiosensitivity by delaying DNA repair, as indicated by the persistence of γ-H2AX foci up to four days post-irradiation. Mechanistically, Oxamate suppressed the expression and phosphorylation of key DNA repair factors. Furthermore, Oxamate induced apoptosis and promoted cellular senescence, as evidenced by the accumulation of SA-β-gal and the upregulation of pS15-p53 and p21. In addition, Oxamate downregulated EGFR expression, reduced the levels of stem cell markers, and modulated epithelial-mesenchymal transition (EMT) markers, suggesting a potential suppression of EMT-related pathways. Together, these results demonstrate that Oxamate enhances radiosensitivity in glioblastoma cells through multiple mechanisms, including the inhibition of DNA repair, induction of apoptosis and senescence, and suppression of cancer stem cell properties and EMT. Our findings provide new insights into the potential use of Oxamate as a radiosensitizer and warrant further investigation of its clinical application in glioblastoma therapy.

Abstract P5-06-06: The Mechanistic Role of BRCA1 DNA and RAD51 Binding in DNA Double-Strand Break Repair

2025-06-13

Angela M. Jasper , Hoang Dinh , Cody M. Rogers +5 more | Clinical Cancer Research

Abstract Genomic instability is a hallmark of cancer, enabling the generation of mutations and gross chromosomal rearrangements to drive neoplastic cell transformation and oncogenesis. The BRCA1-BARD1 protein complex acts to … Abstract Genomic instability is a hallmark of cancer, enabling the generation of mutations and gross chromosomal rearrangements to drive neoplastic cell transformation and oncogenesis. The BRCA1-BARD1 protein complex acts to eliminate highly toxic DNA double-strand breaks, to ensure the faithful propagation of our genetic blueprint and to suppress cancer development. BRCA1 is a well-described tumor suppressor protein associated with hereditary breast and ovarian cancers as well as sporadic breast cancers, with loss or mutation of BRCA1 leading to triple negative breast cancer and poor patient prognosis. The BRCA1-BARD1 complex promotes homologous recombination (HR), which is the major pathway for the accurate repair of double-strand breaks. However, there is little information regarding the intricate roles fulfilled by BRCA1-BARD1 in this process, or how loss of specific BRCA1-BARD1 functions leads to tumorigenesis. BRCA1 has been previously reported to physically interact with both DNA and RAD51, key factors in HR, but the contributions of the interaction attributes to DNA damage repair remain unknown. Here, we delineate major sites of DNA and RAD51 binding in BRCA1 and use a combination of biochemical and NMR methods to identify the specific residues mediating interactions with these ligands. This has allowed us to develop mutations to ablate BRCA1’s ability to interact with these substrates without affecting BRCA1-BARD1’s interaction with other key DNA repair substrates. Using these BRCA1 mutants impaired for either DNA or RAD51 binding, we have interrogated the contributions of these interaction attributes to BRCA1-BARD1’s function by comparing the activity of WT to mutant BRCA1-BARD1 in biochemical assays to reconstitute various steps of HR. We have found that both DNA and RAD51 binding are indispensable for BRCA1-BARD1’s ability to promote RAD51-mediated D-loop formation, thus helping to delineate the mechanism by which BRCA1 promotes HR. Our studies provide the foundation to determine the functional consequences of cancer mutations in BRCA1-BARD1 and for the development of therapeutic strategies to target HR-deficient tumors. Citation Format: Angela Jasper, Hoang Dinh, Cody M. Rogers, Sameer Salunkhe, Hardeep Kaur, Antoine Baudin, David S. Libich, Patrick Sung. The Mechanistic Role of BRCA1 DNA and RAD51 Binding in DNA Double-Strand Break Repair [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-06.

TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

2025-06-13

Jingying Wang , Xia Li , Yuan Qin +1 more | Journal of Translational Medicine

Ophthalmic and Cutaneous Manifestation of Xeroderma Pigmentosum in a 21-Year-Old Man: A Case Report

2025-06-13

Berta Moreno‐Küstner , Moises Lutwak , Stanley Skopit | Cureus

Evolution of TROP2: Biological Insights and Clinical Applications

2025-06-13

Xinyue Zhang , Hong Xiao , Fangjian Na +6 more | European Journal of Medicinal Chemistry

Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein that is overexpressed in a wide range of cancers and is associated with poor prognosis and aggressive tumor behavior. This … Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein that is overexpressed in a wide range of cancers and is associated with poor prognosis and aggressive tumor behavior. This review synthesizes current research on the function of TROP2 in cancer progression through bibliometric analysis, with a special emphasis on advances in the study of antibody-drug conjugates (ADCs) targeting TROP2. We examine the efficacy of targeted therapies against TROP2, in particular the use of sacituzumab govitecan (SG), and evaluate their clinical impact on the treatment of various cancers. Through recent studies and clinical trials, we analyze the clinical effects of TROP2-ADCs and explored the prospects for their application in different cancers, including combination therapies with other therapeutic approaches, studies of resistance mechanisms, and further exploration of TROP2 as a biomarker. This review aims to provide a concise overview of TROP2 applications in cancer therapy, emphasizing emerging trends and therapeutic strategies to provide direction for future research and clinical practice.

Abstract P5-06-26: APOBEC3B And DNA Damage Repair As Synthetic Lethal Pairs

2025-06-13

Bojana Stefanovska , Benjamin Troness , Kevin Lin +2 more | Clinical Cancer Research

Abstract APOBEC3 (A3) enzymes play a pivotal role in mutagenesis across various cancer types. These enzymes, which are single-stranded DNA cytosine deaminases, convert cytosine to uracil (C-to-U) as part of … Abstract APOBEC3 (A3) enzymes play a pivotal role in mutagenesis across various cancer types. These enzymes, which are single-stranded DNA cytosine deaminases, convert cytosine to uracil (C-to-U) as part of innate antiviral immune responses. Aberrant expression of A3 enzymes in several cancers leads to DNA damage, mutagenesis, and genomic instability. In breast cancer, in particular, APOBEC3B (A3B) is the major endogenous mutator that contributes to tumor evolution and resistance to treatment (1–4). DNA C-to-U deamination events result in characteristic C-to-T transitions and C-to-G transversions, and genomic uracils can also be processed into single- or double-stranded DNA breaks and larger chromosomal aberrations. Consequently, tumors with elevated A3B levels endure chronic genotoxic stress and may exhibit increased sensitivity to inhibitors targeting specific DNA repair pathways. Previous research from our laboratory identified DNA uracil glycosylase 2 (UNG2), a critical initiator of the base excision repair (BER) pathway, as a synthetic lethal partner with A3B (5). Genetic disruption of UNG2, combined with high A3B expression, led to cell death (5). This finding suggests that other DNA repair proteins could potentially serve as synthetic lethal partners with A3B under conditions of elevated A3B-induced DNA damage. To test this hypothesis, we used pharmacological and genetic inhibition of DNA damage repair proteins in isogenic cell line models for A3B. Specifically, we quantified cancer cell viability following treatment with various commercially available DNA damage response inhibitors, in the presence or absence of A3B. Additionally, we performed CRISPR screens using a guide RNA library targeting 237 DNA damage repair and response genes in a doxycycline-inducible TREX-293-A3Bi-eGFP cell line. Cells expressing or lacking A3B were harvested for DNA extraction and sequencing at different time points. By comparing guide RNA abundance between doxycycline-treated cells and untreated cells, we identified dropout guides that disrupt genes, creating potential synthetic lethal combinations with A3B. The results from both pharmacological and genetic inhibition approaches will be presented. Selected References: 1. Burns, M. B. et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370 (2013). 2. Law, E. K. et al. The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer. Sci Adv 2, e1601737 (2016). 3. Bertucci, F. et al. Genomic characterization of metastatic breast cancers. Nature 569, 560–564 (2019). 4. Venkatesan, S. et al. Induction of APOBEC3 exacerbates DNA replication stress and chromosomal instability in early breast and lung cancer evolution. Cancer Discov (2021) doi:10.1158/2159-8290.CD-20-0725. 5. Serebrenik, A. A. et al. The deaminase APOBEC3B triggers the death of cells lacking uracil DNA glycosylase. Proc Natl Acad Sci USA 116, 22158–22163 (2019). Citation Format: Bojana Stefanovska, Benjamin Troness, Kevin Lin, Chad Myers, Reuben S. Harris. APOBEC3B And DNA Damage Repair As Synthetic Lethal Pairs [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-26.

Abstract P2-11-10: Assessing the Differential Response to Immunotherapy in BRCA1/2 Mutation Carriers with Triple-Negative Breast Cancer

2025-06-13

Minna Lee , Natalia Polidorio Machado , Varadan Sevilimedu +6 more | Clinical Cancer Research

Abstract Background: The addition of pembrolizumab to standard neoadjuvant chemotherapy (NAC+P) for patients with triple-negative breast cancer (TNBC) has been shown to result in higher rates of pathologic complete response … Abstract Background: The addition of pembrolizumab to standard neoadjuvant chemotherapy (NAC+P) for patients with triple-negative breast cancer (TNBC) has been shown to result in higher rates of pathologic complete response (pCR) and longer event-free survival compared to neoadjuvant chemotherapy alone (NAC). Alterations in the DNA damage response and homologous recombination pathways may differentially affect the tumor-immune microenvironment and therefore influence response to immunotherapy. It is not known whether BRCA1/2 mutation carriers with TNBC have a differential response to immunotherapy compared to noncarriers. Our objective was to compare pCR rates between BRCA1/2 carriers and noncarriers with TNBC undergoing NAC compared to NAC+P. Methods: 341 women with stage II-III TNBC treated with NAC+P based on the KEYNOTE 522 (KN522) regimen from 8/2021-5/2024 were compared to 433 consecutive patients who received NAC prior to 7/2021. Demographic and clinicopathological characteristics, and BRCA1/2 mutational status were collected. pCR rates, defined as no disease in the breast and axilla (ypT0/is pN0), were compared between BRCA1/2 carriers and noncarriers across treatment regimens using Chi square tests. Logistic regression was used to evaluate the association between BRCA1/2 status and pCR. Results: Of non-KN522 patients, 76 (18%) were BRCA1/2 carriers and of KN522 patients, 56 (16%) were BRCA1/2 carriers. Overall, BRCA1/2 carriers were younger, had a lower BMI), and were more likely to be White. There was no difference in histology, grade, presence of lymphovascular invasion, clinical T or N stage, or chemotherapy regimen between carriers and noncarriers. The pCR rates in the non-KN522 group were 50% for BRCA1/2 carriers compared to 36% for noncarriers (p=0.021). In the KN522 group, pCR rates were 68% for BRCA1/2 carriers compared to 55% for noncarriers (p=0.073). Among mutation carriers treated with KN522, the pCR rate was 69% (31/45) for BRCA1 and 60% (6/10) for BRCA2 carriers. On multivariable analysis for the entire cohort, BRCA1/2 status (OR 1.55, 95% CI 1.03-2.34, p=0.036), receipt of KN522 (OR 2.13, 95% CI 1.71-3.12, p&lt;0.001), younger age at diagnosis (OR 0.98, 95% CI 0.97-0.99, p=0.002), and high grade (OR 9.42, 95% CI 4.01-27.6, p&lt;0.001) were associated with pCR. Among patients receiving KN522, only high grade was associated with pCR (OR 17.4, 95% CI 5.03-110, p&lt;0.001); BRCA1/2 status, age, BMI, race, and stage were not associated with pCR. Conclusion: BRCA1/2 carriers with TNBC had better response to NAC compared to noncarriers. This was also observed in patients who received NAC+P, although this did not reach statistical significance. Additional work with a larger sample size of carriers may further delineate the differential response to NAC+P. This will be increasingly important for BRCA1/2 carriers as we investigate optimal escalation and de-escalation strategies, particularly when immunotherapy is combined with alternative therapies (i.e., PARP inhibitors). Citation Format: Minna Lee, Natalia Polidorio Machado, Varadan Sevilimedu, Lauren Perry, Giacomo Montagna, Monica Morrow, Nour Abuhadra, George Plitas, Stephanie Downs-Canner. Assessing the Differential Response to Immunotherapy in BRCA1/2 Mutation Carriers with Triple-Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-11-10.

Abstract P5-04-14: Variants of PEAK1, SLC2A4RG, PALB2, and XIRP2 and their relationship with Genetic Ancestry across Breast Cancer Patients from different Racial/Ethnic groups

2025-06-13

Gloria Crespo , Julie Dutil , Vivian Colón López | Clinical Cancer Research

Abstract Breast cancer is the second leading cause of cancer deaths worldwide, affecting 1 in 8 women. Disparities in breast cancer incidence and mortality have been observed across different racial … Abstract Breast cancer is the second leading cause of cancer deaths worldwide, affecting 1 in 8 women. Disparities in breast cancer incidence and mortality have been observed across different racial and ethnic groups, with Hispanic and African American women experiencing higher incidence and mortality rates. Differences in the genomic landscape of breast cancer patients have been linked to early age at diagnosis, worse disease progression, drug resistance, and relapse. Using the All of Us research hub, one of the largest biomedical databases, we are investigating genetic variants and the genetic ancestry related to PEAK1, PALB2, SLC2A4RG, and XIRP2 across racial and ethnic groups to identify potential variations that can contribute to differences observed among these groups. These genes are involved in DNA repair, epithelial-to-mesenchymal transition (EMT), and cell cycle regulation. PEAK1, for instance, regulates cell proliferation, promotes invasion and metastasis in various cancers, including breast cancer, and is associated with drug resistance. PALB2 functions as a tumor suppressor involved in homologous recombination repair, with pathogenic variants linked to hereditary breast cancer in Hispanics. SLC2A4RG acts as a tumor suppressor by regulating the cell cycle and has been studied in glioblastoma and breast cancer. XIRP2, known for its roles in cardiovascular diseases, is implicated in breast cancer metastasis and colon cancer promotion. Despite identifying many genes involved in breast cancer development and metastasis, there remains a need to understand genetic variability across different populations to develop better diagnostic tools and improve treatment for breast cancer patients. Our cohort includes 6,056 cases of breast cancer, from which our preliminary results showed that Hispanics and African Americans are diagnosed with breast cancer at an earlier age (median = 64 and 67 years, respectively) compared to Non-Hispanics (median = 71 years). Using a genetic ancestry approach and in accordance with other studies, we identified that the majority of our Hispanic population are related to the Admixed American ancestry, with smaller portions of European and African ancestry. By accessing the All of Us research hub's control tier for genetic data, we identified the frequency of genetic variants of PEAK1, PALB2, SLC2A4RG, and XIRP2. Using a Minor Allele Frequency (MAF) of 0.01 as a parameter, we identified 27 variants for PEAK1, 12 for PALB2, 2 for SLC2A4RG, and 99 for XIRP2. Our current and upcoming analyses include associating the identified genetic variants with ancestry and race/ethnicity among cases already identified in our cohort and dataset, and comparing them with our control groups to identify possible genetic variants involved in breast cancer development across the different populations under study. Citation Format: Grace Vélez Crespo, Julie Dutil, Vivian Colón López. Variants of PEAK1, SLC2A4RG, PALB2, and XIRP2 and their relationship with Genetic Ancestry across Breast Cancer Patients from different Racial/Ethnic groups [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-04-14.

Abstract P1-08-27: Unusual Presentation Of Triple-Negative Breast Cancer (TNBC) In A Young Female Patient With Xeroderma pigmentosum (XP)

2025-06-13

Bashaer Alsaati , Nora Trabulsi , Reem Ujami +1 more | Clinical Cancer Research

Abstract Background: Xeroderma pigmentosum (XP) is a rare, autosomal recessive (1 in a million) disorder characterized by sensitivity to ultraviolet (UV) rays, resulting in a predisposition to skin cancers, primarily. … Abstract Background: Xeroderma pigmentosum (XP) is a rare, autosomal recessive (1 in a million) disorder characterized by sensitivity to ultraviolet (UV) rays, resulting in a predisposition to skin cancers, primarily. This case vignette describes an unusual presentation of triple-negative breast cancer (TNBC) in a young female patient with XP. Case Presentation: A 34-year-old woman with a known history of (XP) presented with a palpable mass in her right breast. Clinical examination revealed a firm, irregular, and non-tender mass in the upper outer quadrant of the right breast, measuring approximately 6 cm in diameter. There was no axillary lymphadenopathy. Mammography and ultrasound confirmed a suspicious lesion, and a core needle biopsy was performed. No family history of breast cancer or other malignancies and gBRCA1&2 were negative. Histopathological examination of the biopsy specimen revealed grade III invasive ductal carcinoma, negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), consistent with TNBC. Management and Outcome: A neoadjuvant chemotherapy (NACT) with four cycles of doxorubicin and cyclophosphamide (AC) DD was prescribed. The response to NACT was limited. Consequently, the patient underwent surgery with a modified radical mastectomy (MRM) with tumor of 6 cm & sentinel lymph node biopsy was negative for metastasis. Postoperative follow-up included adjuvant CT with paclitaxel plus carboplatin. Immunotherapy was not accessible for her. Adjuvant radiotherapy was not prescribed. Discussion: 1.Association of XP and BC: The evidence that XP patients may also have an elevated risk of internal malignancies is scarce. Understanding the mechanisms linking XP with breast cancer, such as DNA repair deficiencies and genomic instability, could provide insights into targeted prevention and treatment strategies. 2.Addition of Carboplatin in Adjuvant Chemotherapy: Studies suggest that the inclusion of carboplatin in the NACT for TNBC can improve pathological complete response rates and survival outcomes. Given the limited response to initial chemotherapy in this patient, the addition of adjuvant carboplatin was commenced to enhance treatment efficacy and potentially reduce the risk of recurrence. 3.Consideration of Adjuvant Radiotherapy (XRT) in XP Patients: The use of XRT in XP patients is challenging due to their heightened sensitivity to UV and ionizing radiation. Protective measures and alternative modalities should be explored to minimize potential adverse effects. Our patient was offered MRM in an attempt to avoid XRT. 4. Type of Surgery: MRM vs. Breast-Conserving Surgery (BCS) : The choice between MRM and BCS in XP patients with TNBC must consider oncologic outcomes and future potential risk of XRT, especially the long -term safety of XRT in such patient is questionable. Conclusion: This case highlights the complexity of managing TNBC in patients with XP. It underscores the need for personalized treatment plans that address the unique challenges posed by XP. Multidisciplinary collaboration and further research are essential to optimize therapeutic strategies for these high-risk patients. Citation Format: Bashaer Alsaati, Nora Trabulsi, Reem Ujami, Atlal Abusanad. Unusual Presentation Of Triple-Negative Breast Cancer (TNBC) In A Young Female Patient With Xeroderma pigmentosum (XP) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-08-27.

Abstract P1-05-08: Germline Mutations in Early Triple-Negative Breast Cancer Brazilian Patients

2025-06-13

Gerardo Zanetta , I. Ferreira , Helton C. Santiago +7 more | Clinical Cancer Research

Abstract Background: Triple-negative breast cancer (TNBC) is pathologically defined by the absence of hormonal receptors and HER2 expression. This subtype accounts for approximately 20% of all breast cancers and is … Abstract Background: Triple-negative breast cancer (TNBC) is pathologically defined by the absence of hormonal receptors and HER2 expression. This subtype accounts for approximately 20% of all breast cancers and is known for its worse prognosis. The literature demonstrates a high association of TNBC with germline mutations in BRCA1 and other genes. With the development of targeted therapies, this information impacts the therapeutic decision for these patients. Methods: Retrospective, single-institutional, descriptive study, which recruited consecutive patients submitted to neoadjuvant chemotherapy for early TNBC between 2010 and 2023. Data was retrieved from medical records. The primary objective was to describe germline hereditary breast and ovarian cancer (HBOC) test results in this population. Results: 394 TNBC patients were included. The median age was 44, and most were premenopausal women (64%). Two hundred sixty-two patients (66.5%) had a positive family history of cancer, and 242 (61.4%) underwent a germline HBOC test. A germline mutation was present in 128 patients (52.9% of the tests): 24.4% of tested patients (15% of all patients) had variants of uncertain significance (VUS) and 31% of pathogenic mutations (19% of all patients). The most prevalent VUS were in BRCA2 (N=9; 3.7%), ATM (N=8; 3.3%), BRCA1 (N=6 2.5%), and mismatch-repair genes (N=9; 3.7%). The pathogenic mutations prevailed in BRCA1 (N=45; 18.6%), BRCA2 (N=9; 3.7%), and RAD15C (N=6; 2.5%). Together, germline pathogenic mutations in BRCANESS genes were present in 68 patients (28.1% of all tests). Two patients had co-pathogenic mutations in CHEK2 and TP53. Considering the tested cohort, 21.1% of &gt; 60-year-old patients and 27.8% of patients without a family history of cancer presented pathogenic germline mutations. Conclusion: The high prevalence of pathogenic mutations in this cohort highlights the importance of germline tests and genetic counseling for TNBC patients, despite age or family history criteria. Citation Format: Giulia Zanetta, Giulia K. Zanetta, Izabela P. Ferreira, Hendrio R. Santiago, Flavia C. Balint, Monique C. Tavares, Luciana M. Leite, Felicia P. Cavalher, Solange M. Sanches, Vladmir C. C. Lima, Marcelle G. Cesca. Germline Mutations in Early Triple-Negative Breast Cancer Brazilian Patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-08.

Loss of meiotic double strand breaks triggers recruitment of recombination-independent pro-crossover factors in C. elegans spermatogenesis

2025-06-13

JoAnne Engebrecht , Aashna Calidas , Qianyan Li +5 more | bioRxiv (Cold Spring Harbor Laboratory)

A key event in meiosis is the conversion of a small subset of double strand breaks into interhomolog crossovers. In this study, we demonstrate that Caenorhabditis elegans male spermatogenesis has … A key event in meiosis is the conversion of a small subset of double strand breaks into interhomolog crossovers. In this study, we demonstrate that Caenorhabditis elegans male spermatogenesis has less robust mechanisms than hermaphrodite oogenesis for ensuring and limiting the conversion of double strand breaks into crossovers. This is not a consequence of differences in meiotic prophase timing, sex chromosome genotype, or the presence or absence of germline apoptosis. Using the cyclin-like crossover marker COSA-1, we show that males have a linear response in converting increasing numbers of double strand breaks into crossovers, suggesting weakened crossover homeostasis. While the topoisomerase SPO-11, responsible for initiating meiotic double strand breaks, has an extended period of activity in males as in hermaphrodites, we discovered that COSA-1 foci form at the very end of meiotic prophase in the absence of SPO-11 during spermatogenesis. These COSA-1-marked sites are also independent of homologous recombination, and Topoisomerases I and II. We find that the synaptonemal complex, which holds homologs in proximity, differently modulates COSA-1 enrichment to chromosomes in the absence of SPO-11 in males and hermaphrodites. Together, these findings suggest that males have less robust crossover control and that there are previously unrecognized lesions or structures at the end of meiotic prophase in spermatocytes that can accumulate CO markers.

Integrating breast tumor homologous recombination deficiency status to aid germline BRCA1 and BRCA2 variant classification

2025-06-13

Cristina Fortuño , Jia Zhang , Lambros T. Koufariotis +7 more | medRxiv (Cold Spring Harbor Laboratory)

Pathogenic germline variants in certain genes are associated with somatic tumor mutation signatures. The use of somatic tumor mutation data has the potential to improve the identification of true pathogenic … Pathogenic germline variants in certain genes are associated with somatic tumor mutation signatures. The use of somatic tumor mutation data has the potential to improve the identification of true pathogenic variants but remains underexplored. We investigated the integration of tumor homologous recombination (HR) deficiency status as a predictor of pathogenicity for germline BRCA1 and BRCA2 variants, building on the established link between HR deficiency and germline pathogenic variants in these genes. We analyzed breast tumor whole-genome sequence and matching germline data from 350 patients across four datasets: Familial Breast Cancer (N=77), The Cancer Genome Atlas (TCGA-BRCA, N=96), the MAGIC study (N=136), and Q-IMPROvE (N=41). A total of 13,364 germline variants (including structural variations) in BRCA1, BRCA2, and other DNA repair genes (ATM, BARD1, BRIP1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, TP53) underwent variant curation. Patients were categorized based on germline classification as BRCA1 positive (N=25), BRCA2 positive (N=21), and BRCA1/2 negative (N=149), excluding those with BRCA1/2 variants of uncertain significance (N=21) and pathogenic variants in other DNA repair genes (N=134). Somatic HR status (deficient or proficient) was predicted using three algorithms: HRDetect, CHORD, and HRDSum. HR-deficient and HR-proficient status were significant predictors of germline BRCA1/2 pathogenic variant status (positive and negative directions). The CHORD algorithm, which estimates BRCA1 and BRCA2 subtype specifically, added precision contributing evidence towards pathogenicity for the corresponding gene. Finally, we assessed CHORD HR predictions for variants of uncertain significance in BRCA1 and BRCA2, and reported their tumor HR status for potential use as additional evidence in variant curation. Analysis across multiple tumor whole-genome sequencing datasets has shown that HR status prediction algorithms can separate profiles for BRCA1 and BRCA2 pathogenic variants and provide further evidence at increased weight to aid in the classification of germline BRCA1 and BRCA2 variants. Tumor sequencing offers a promising strategy for reducing the uncertainty in germline variant interpretation.

Targeting DNA damage in ageing: towards supercharging DNA repair

2025-06-12

Arturo Bujarrabal , George A. Garinis , Paul D. Robbins +2 more | Nature Reviews Drug Discovery

DNA Damage Repair Pathway Alterations and Immune Landscape Differences in Pediatric/Adolescent, Young Adult (AYA) and Adult Sarcomas

2025-06-12

Kurt Statz-Geary , Andrew Elliott , Steven Bialick +7 more | Cancers

Background: DNA damage response (DDR) pathway alterations contribute to genomic instability and malignant progression in several cancers. Methods: We retrospectively reviewed molecular profiles from 5309 sarcoma patient samples, including 746 … Background: DNA damage response (DDR) pathway alterations contribute to genomic instability and malignant progression in several cancers. Methods: We retrospectively reviewed molecular profiles from 5309 sarcoma patient samples, including 746 from pediatric/adolescent and young adults (ped/AYA), encompassing 38 histologic subtypes. The gene expression profiles were further analyzed for immunotherapy-related biomarker associations, including analysis of the T cell-inflamed score. Results: Pathogenic/likely pathogenic DDR alterations were detected in 15.9% (N = 842) of samples overall and 9.25% (N = 69) of Ped/AYA tumors, with mutations occurring most frequently in ATRX (10.1%). Shorter overall survival was observed for patients with DDR-alterations compared to those with DDR-wildtype tumors (Hazard Ratio = 1.172, 95% CI: 1.068–1.287; p < 0.001). In many subtypes, DDR-mutated tumors were found to have increased rates of immune markers, including PD-L1+, dMMR/MSI-high, and TMB. Conclusions: Our study of somatic DDR-pathway mutations provides a better understanding of the molecular associations across sarcoma subtypes that may aid in developing future prognostic and therapeutic options for these rare cancers.