ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair.
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ABSTRACT:
SUBMITTER: Segal-Raz H
PROVIDER: S-EPMC3128972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with ?-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.
| S-EPMC4300955 | biostudies-literature
Project description:APLF is a forkhead associated-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser(116)). Here, we demonstrate that the phosphorylation of APLF at Ser(116) in human U2OS cells by ATM is dependent on poly(ADP-ribose) polymerase 3 (PARP3) levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser(116) to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the level of accumulation of APLF at sites of laser-induced DNA damage and impaired the accumulation of Ser(116)-phosphorylated APLF at IR-induced ?H2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signalling pathway to facilitate APLF-Ser(116) phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that Ser(116)-APLF phosphorylation facilitates APLF-dependent double-strand break repair.
| S-EPMC3627606 | biostudies-literature
Project description:The chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5' end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage.
| S-EPMC5993204 | biostudies-literature
Project description:DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and approximately 4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.
| S-EPMC3071034 | biostudies-literature
Project description:The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.
| S-EPMC3624173 | biostudies-other
Project description:The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.
| S-EPMC126026 | biostudies-literature
Project description:In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.
| S-EPMC3213282 | biostudies-literature
Project description:The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2- and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.
| S-EPMC343805 | biostudies-other
Project description:Protein phosphatase 2A (PP2A) functions as a potent tumor suppressor, but its mechanism(s) remains enigmatic. Specific disruption of PP2A by either expression of SV40 small tumor antigen or depletion of endogenous PP2A/C by RNA interference inhibits Ku DNA binding and DNA-PK activities, which results in suppression of DNA double-strand break (DSB) repair and DNA end-joining in association with increased genetic instability (i.e., chromosomal and chromatid breaks). Overexpression of the PP2A catalytic subunit (PP2A/C) enhances Ku and DNA-PK activities with accelerated DSB repair. Camptothecin-induced DSBs promote PP2A to associate with Ku 70 and Ku 86. PP2A directly dephosphorylates Ku as well as the DNA-PK catalytic subunit (DNA-PKcs) in vitro and in vivo, which enhances the formation of a functional Ku/DNA-PKcs complex. Intriguingly, PP2A promotes DSB repair in wild type mouse embryonic fibroblast (MEF) cells but has no such effect in Ku-deficient MEF cells, suggesting that the Ku 70/86 heterodimer is required for PP2A promotion of DSB repair. Thus, PP2A promotion of DSB repair may occur in a novel mechanism by activating the nonhomologous end-joining pathway through direct dephosphorylation of Ku and DNA-PKcs, which may contribute to maintenance of genetic stability.
| S-EPMC2745667 | biostudies-literature
Project description:Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.
| S-EPMC3569989 | biostudies-literature