Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the S_N2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PK_CS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S_N2-alkylating agents.     

Topoisomerase II-Mediated DNA Damage Is Differently Repaired during the Cell Cycle by Non-Homologous End Joining and Homologous Recombination

Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2α was largely responsible for the induction of γH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells.     

与细胞周期G2/M期进程相关的H2AX磷酸化

γH2AX焦点(foci)被普遍当做DNA双链断裂（DSB）损伤的分子标志物.为探 讨细胞周期进程相关的H2AX磷酸化规律特征,采用胸腺嘧啶双阻滞结合噻氨酯哒唑(nocodazole)的后续处理,将HeLa细胞同步于有丝分裂的前中期．然后,用流式细胞仪检测细胞周期、Western印迹和免疫荧光法,观察γH2AX表达和γH2AX焦点的形成．结果显示,细胞进入G2/M期和有丝分裂过程中,γH2AX水平显著增加 ;在无DNA DSB发生的情况下,部分M期细胞中也存在大量的γH2AX焦点．随着细 胞完成有丝分裂从M期退出再进入G1期,γH2AX的表达水平逐渐降低．这种 γH2AX表达变化特征与G2/M期密切关联的PLK1和Cyclin B1的表达规律相类似． 在4 Gy大剂量照射下,HeLa细胞于照后8 到12 h出现明显的G2/M期阻滞．γH2AX 焦点数在照后1 h达高峰,随后降低,照后8 h又上升,出现了第2个峰值．与之不同的是,在1 Gy低剂量照射下,细胞的G2/M期阻滞微弱,γH2AX焦点数在照后 0.5 h最高,随后下降,且无反弹,符合DNA DSB的修复动力学特征．因此,将γ H2AX当做DNA DSB分子标志物时,还需要考虑细胞周期变化的影响．γH2AX适合 作为1 Gy以下照射的DNA双链断裂损伤的分子标志．     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Histone deacetylase inhibitor sodium butyrate enhances cellular radiosensitivity by inhibiting both DNA nonhomologous end joining and homologous recombination

HDAC inhibitors have been proposed as radiosensitizers in cancer therapy. Their application would permit the use of lower radiation doses and would reduce the adverse effects of the treatment. However, the molecular mechanisms of their action remain unclear. In the present article, we have studied the radiosensitizing effect of sodium butyrate on HeLa cells. FACS analysis showed that it did not abrogate the γ-radiation imposed G2 cell cycle arrest. The dynamics of γ-H2AX foci disappearance in the presence and in the absence of butyrate, however, demonstrated that butyrate inhibited DSB repair. In an attempt to clarify which one of the two major DSBs repair pathways was affected, we synchronized HeLa cells in G1 phase and after γ-irradiation followed the repair of the DSBs by agarose gel electrophoresis. Since HR is not operational during G1 phase, by this approach we determined the rates of NHEJ only. The results showed that NHEJ decreased in the presence of butyrate. In another set of experiments, we followed the dynamics of disappearance of RAD51 foci in the presence and in the absence of butyrate after γ-radiation of HeLa cells. Since RAD51 takes part in HR only, this experiment allows the effect of butyrate on DSB repair by homologous recombination to be assessed. It showed that HR was also obstructed by butyrate. These results were confirmed by host cell reactivation assays in which the repair of plasmids containing a single DSB by NHEJ or HR was monitored. We suggest that after a DSB is formed, HDACs deacetylated core histones in the vicinity of the breaks in order to compact the chromatin structure and prevent the broken DNA ends from moving apart from each other, thus ensuring effective repair.     

The Efficiency of Homologous Recombination and Non-Homologous End Joining Systems in Repairing Double-Strand Breaks during Cell Cycle Progression

This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5–24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.     

Activation of ATM/ATR signaling in human embryonic stem cells after DNA damage

Embryonic stem cells (ESCs) are the progenitors of all adult cells; consequently, genomic abnormalities in them may be catastrophic for the developing organism. ESCs are characterized by high proliferation activity and do not stop in checkpoints upon DNA-damage executing only G₂/M delay after DNA damage. ATM and ATR kinases are key sensors of double-strand DNA breaks and activate downstream signaling pathways involving checkpoints, DNA repair, and apoptosis. We examined activation of ATM/ATR signaling in human ESCs and revealed that irradiation induced ATM, ATR, and Chk2 phosphorylation, and γH2AX foci formation and their colocalization with 53BP1 and Rad51 proteins. Interestingly, human ESCs exhibit noninduced γH2AX foci colocalized with Rad51 and marking single-strand DNA breaks. Next, we revealed the significant contribution of ATM, Chk1, and Chk2 kinases to G₂/M block after irradiation and ATM-dependent activation (phosphorylation) of p53 in human ESCs. However, p53 activation and subsequent induction of p21 ^Waf1 gene expression after DNA damage do not result in p21^Waf1 protein accumulation due to its proteasomal degradation.     

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by gammaH2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced DSB in mammalian cells.     

Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.     

Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity

The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC₅₀ in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.     

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.     

γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths,limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating γH2AX foci that can be visualised by immunofluorescence. There is a close correlation between γH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, γH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionising radiation, γH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimise the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.     

The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells

DNA polymerase (Pol) β null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol β, SSB accumulate in G₁ phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol β null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.     

Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.     

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.     

γH2A binds Brc1 to maintain genome integrity during S‐phase

ATM^Tel1 and ATR^Rad3 checkpoint kinases phosphorylate the C‐terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho‐H2A/X (γH2A/X)‐binding proteins at double‐strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X‐ray scattering (SAXS), and X‐ray structural studies of the Schizosaccharomyces pombe Brc1, a 6‐BRCT‐domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds γH2A to form spontaneous and DNA damage‐induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage‐induced Brc1 foci colocalize with DSB response factors. γH2A binding is critical for Brc1 function. The 1.45 Å resolution crystal structure of Brc1–γH2A complex shows how variable BRCT insertion loops sculpt tandem‐BRCT phosphoprotein‐binding pockets to facilitate unique phosphoprotein‐interaction specificities, and unveils an acidic DNA‐mimicking Brc1 surface. From these results, Brc1 docking to γH2A emerges as a critical chromatin‐specific response to replication‐associated DNA damage.