Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.     

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.     

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.     

Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.Key words: protein phosphatase, PP6, γ-H2AX, DNA double-strand break, homology-directed repair     

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.     

Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.     

MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways

Purpose: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines.Methods: Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting.Results: GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1.Conclusion: GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.     

Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.     

USP11 Is a Negative Regulator to γH2AX Ubiquitylation by RNF8/RNF168

Ubiquitin modification at double strand breaks (DSB) sites is an essential regulator of signaling and repair. γH2AX extends from DSB sites and provides a platform for subsequent recruitment and amplification of DNA repair proteins and signaling factors. Here, we found that RNF8/RNF168 ubiquitylates γH2AX. We identified that USP11 is a unique deubiquitylation enzyme for γH2AX. USP11 deubiquitylates γH2AX both in vivo and in vitro but not the canonical (ub)-K119-H2A and (ub)-K120-H2B in vitro, and USP11 ablation enhances the levels of γH2AX ubiquitylation. We also found that USP11 interacts with γH2AX both in vivo and in vitro. We found that 53BP1 and ubiquitin-conjugated proteins are misregulated to be retained longer and stronger at DSB sites after knockdown of USP11. We further found that cells are hypersensitive to γ-irradiation after ablation of USP11. Together, our findings elucidate deeply and extensively the mechanism of RNF8/RNF168 and USP11 to maintain the proper status of ubiquitylation γH2AX to repair DSB.     

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.     

High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.     

Accumulation of DSBs in gamma-H2AX domains fuel chromosomal aberrations

DNA double strand breaks (DSBs) pose a severe hazard to the genome as erroneous rejoining of DSBs can lead to mutation and cancer. Here, we have investigated the correlation between X irradiation-induced γ-H2AX foci, theoretically induced DSBs, and the minimal number of mis-rejoined DNA breaks (MNB) in irradiated lymphocytes obtained from two healthy humans by painting of the whole chromosome complement by spectral karyotyping. There were less γ-H2AX foci/dose than theoretically expected, while misrepair, as expressed by MNB/γ-H2AX focus, was similar at 0.5 and 1 Gy but 3.6-fold up at 3 Gy. Hence, our results suggest that X-ray-induced γ-H2AX foci in G0 lymphocyte nuclei contain more than one DSB and that the increasing number of DSBs per γ-H2AX repair factory lead to an increased rate of misrepair.     

SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks

The gene mutated in Bloom''s syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased γ-H2AX foci. Because the increased γ-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM''s function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess γ-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM''s pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.     

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.     

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study

Background

In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).

Objective

To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection.

Methods

Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.

Results

The median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL.

Conclusion

γ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.     

The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage

Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)–induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with γ-H2AX and ATM. HINT1 deficiency does not affect the formation of γ-H2AX foci; however, it impairs the removal of γ-H2AX foci after DNA damage and this is associated with impaired acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both γ-H2AX and ATM.     

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.     

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.