Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks

Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G₁ cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5′ to 3′ resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G₁, and markedly repressed at G₂. Repression of NHEJ at G₂ is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5′ end resection by CDK1 inhibition at G₂ alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G₁. Expression of excess Ku can partially offset the inhibition of end joining at G₂. The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.     

Metastasis suppressor NME1 promotes non-homologous end joining of DNA double-strand breaks

NME1 (also known as NM23-H1) was the first identified tumor metastasis suppressor, which has been reported to link with genomic stability maintenance and cancer. However its underlying mechanisms are still not fully understood. Here we find that NME1 is required for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Mechanistically, NME1 re-localizes to DNA damage sites in a Ku-XRCC4-dependent manner, and regulates downstream LIG4 recruitment and end joining efficiency. Furthermore, we show that the 3′-5′ exonuclease activity of NME1 is critical for its function in NHEJ. Taken together, our findings identify NME1 as a novel NHEJ factor, and reveal how this metastasis suppressor promotes genome stability.     

Mechanistic analysis of Xenopus EXO1's function in 5'-strand resection at DNA double-strand breaks

The processing of DNA double-strand breaks (DSBs) into 3' single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5'-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis. Xenopus EXO1 (xEXO1) was found to display strong 5'→3' dsDNA exonuclease activity but no significant ssDNA exonuclease activity. Depletion of xEXO1 caused significant inhibition of 5' strand resection. Co-depletion of xEXO1 and Xenopus DNA2 (xDNA2) showed that these two nucleases act in parallel pathways and by distinct mechanisms. While xDNA2 acts on ssDNA unwound mainly by the Xenopus Werner syndrome protein (xWRN), xEXO1 acts directly on dsDNA. Furthermore, xEXO1 and xWRN are required for both the initiation stage and the extension stage of resection. These results reveal important novel information on the mechanism of 5'-strand resection in eukaryotes.     

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.     

The ubiquitin landscape at DNA double-strand breaks

The intimate relationship between DNA double-strand break (DSB) repair and cancer susceptibility has sparked profound interest in how transactions on DNA and chromatin surrounding DNA damage influence genome integrity. Recent evidence implicates a substantial commitment of the cellular DNA damage response machinery to the synthesis, recognition, and hydrolysis of ubiquitin chains at DNA damage sites. In this review, we propose that, in order to accommodate parallel processes involved in DSB repair and checkpoint signaling, DSB-associated ubiquitin structures must be nonuniform, using different linkages for distinct functional outputs. We highlight recent advances in the study of nondegradative ubiquitin signaling at DSBs, and discuss how recognition of different ubiquitin structures may influence DNA damage responses.     

Cell nonhomologous end joining capacity controls SAF-A phosphorylation by DNA-PK in response to DNA double-strand breaks inducers

Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by nonhomologous end joining. These results bring a new link between the hnRNP family and the DNA damage response. Additionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors.     

Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1^Ser25 and 53BP1^Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1^Ser25 and 53BP1^Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.     

Nuclear organization in DNA end processing: Telomeres vs double-strand breaks

Many proteins ligands are shared between double-strand breaks and natural chromosomal ends or telomeres. The structural similarity of the 3’ overhang, and the efficiency of cellular DNA end degradation machineries, highlight the need for mechanisms that resect selectively to promote or restrict recombination events. Here we examine the means used by eukaryotic cells to suppress resection at telomeres, target telomerase to short telomeres, and process broken ends for appropriate repair. Not only molecular ligands, but the spatial sequestration of telomeres and damage likely ensure that these two very similar structures have very distinct outcomes with respect to the DNA damage response and repair.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks.

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

Caffeine-induced radiosensitization is independent of nonhomologous end joining of DNA double-strand breaks

After exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two major complementary DNA double-strand break (DSB) repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). The relationship between checkpoint activation and these two types of DNA DSB repair pathways is not clear. Caffeine, as a nonspecific inhibitor of ATM and ATR, abolishes multi-checkpoint responses and sensitizes cells to radiation-induced killing. However, it remains unknown which DNA repair process, NHEJ or HRR, or both, is affected by caffeine-abolished checkpoint responses. We report here that caffeine abolishes the radiation-induced G(2)-phase checkpoint and efficiently sensitizes both NHEJ-proficient and NHEJ-deficient mammalian cells to radiation-induced killing without affecting NHEJ. Our results indicate that caffeine-induced radiosensitization occurs by affecting an NHEJ-independent process, possibly HRR.     

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.     

Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.     

Capture of extranuclear DNA at fission yeast double-strand breaks

Proper repair of DNA double-strand breaks (DSBs) is necessary for the maintenance of genomic integrity. Here, a new simple assay was used to study extrachromosomal DSB repair in Schizosaccharomyces pombe. Strikingly, DSB repair was associated with the capture of fission yeast mitochondrial DNA (mtDNA) at high frequency. Capture of mtDNA fragments required the Lig4p/Pku70p nonhomologous end-joining (NHEJ) machinery and its frequency was highly increased in fission yeast cells grown to stationary phase. The fission yeast Mre11 complex Rad32p/Rad50p/Nbs1p was also required for efficient capture of mtDNA at DSBs, supporting a role for the complex in promoting intermolecular ligation. Competition assays further revealed that microsatellite DNA from higher eukaryotes was preferentially captured at yeast DSBs. Finally, cotransformation experiments indicated that, in NHEJ-deficient cells, capture of extranuclear DNA at DSBs was observed if homologies--as short as 8 bp--were present between DNA substrate and DSB ends. Hence, whether driven by NHEJ, microhomology-mediated end-joining, or homologous recombination, DNA capture associated with DSB repair is a mutagenic process threatening genomic stability.     

DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

Non-homologous end joining (NHEJ) is the major pathway that mediates the repair of DNA double-strand breaks (DSBs) generated by ionizing radiation (IR). Previously, the DNA helicase RECQL4 was implicated in promoting NHEJ, but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PK_cs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PK_cs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PK_cs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PK_cs is required for accumulation of RECQL4 to DSBs and that DNA-PK_cs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PK_cs in NHEJ.     

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues.