Ku recruits XLF to DNA double-strand breaks

XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.     

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.     

Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal α/β, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

Mammalian cells have an activity of mutagenic repair for DNA double-strand breaks (DSBs), microhomology-mediated end joining (MMEJ), in which DNA ends are joined via microhomologous sequences flanking the breakpoint. MMEJ has been indicated to be undertaken without Ku proteins, which are essential factors for non-homologous end joining (NHEJ). On the other hand, recent studies with cell-free (in vitro) systems indicated the involvement of Ku proteins in MMEJ, suggesting that MMEJ could be also undertaken by a Ku-dependent pathway. To clarify whether Ku proteins are essential in MMEJ in vivo, linearized plasmid DNAs with microhomologous sequences of 10bp at both ends were introduced as repair substrates into Ku80-proficient and Ku80-deficient CHO cells, and were subjected to MMEJ and NHEJ. Activities of MMEJ and NHEJ, respectively, of the cells were evaluated by mathematical modeling for the increase in fluorescence of GFP proteins produced from repaired products. The Ku80 deficiency caused approximately 75% reduction of the MMEJ activity in CHO cells, while it caused is > or =90% reduction of the NHEJ activity. Therefore, it was indicated that there is a Ku-dependent pathway for MMEJ; however, MMEJ is less dependent on Ku80 protein than NHEJ. The fraction of MMEJ products increased in proportion to the increase in the amounts of substrates. The results suggest that the increase in DSBs makes the cell more predominant for MMEJ. MMEJ might function as a salvage pathway for DSBs that cannot be repaired by NHEJ.     

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.     

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.     

Radiation-induced recombination is dependent on Ku80

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.     

Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks

In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.     

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1–BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.     

Role for RIF1-interacting partner DDX1 in BLM recruitment to DNA double-strand breaks

Human Rap1-interacting factor 1 (RIF1) is an important player in the repair of DNA double strand breaks (DSBs). RIF1 acts downstream of 53BP1, with well-documented roles in class switch recombination in B-cells and inhibition of end resection initiation in BRCA1-defective cells. Here, we report that DEAD Box 1 (DDX1), a RNA helicase also implicated in DSB repair, interacts with RIF1, with co-localization of DDX1 and RIF1 observed throughout interphase. Recruitment of DDX1 to DSBs is dependent on RIF1, with RIF1 depletion abolishing DDX1-mediated facilitation of homologous recombination at DSBs. As previously demonstrated for RIF1, DDX1 is also required for chromatin loading of Bloom syndrome helicase (BLM) to ionizing radiation-induced DSBs, a RIF1-related activity that is independent of 53BP1. We show that DDX1 and RIF1 have different nucleic acid requirements for accumulation at DSBs, with RNA-DNA hybrids required for DDX1 accrual at DSBs, and single-strand RNA required for accumulation of RIF1 at these sites. Our data suggest both convergent and divergent roles for DDX1 and RIF1 in DSB repair, and may help explain why RIF1 depletion does not fully mimic 53BP1 ablation in the restoration of homologous recombination defects in BRCA1-deficient cells.     

Recruitment of proteins to DNA double-strand breaks: MDC1 directly recruits RAP80

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. Occurrence of DSBs in the cell activates the DNA damage response (DDR), which involves signaling cascades that sense and respond to the damage. Promptly after DSB induction, DDR proteins accumulate surrounding both DNA ends and form microscopically-visible foci. Recently, we demonstrated that the key DDR protein MDC1 directly binds RAP80, an additional DDR protein that recruits BRCA1 to DSBs. We provided evidences that the MDC1-RAP80 interaction depends on a ubiquitylation event on K-1977 of MDC1. However, it remained unknown whether K-1977 of MDC1 is required for the recruitment of RAP80 to DSBs. Here we show that K-1977 of MDC1 is necessary for focus formation by RAP80. Nevertheless, it has not effect on focus formation by γ-H2AX, MDC1 or 53BP1. The results imply a role for the MDC1-RAP80 interaction in focus formation by the RAP80-BRCA1 complex. In light of these recent results we discuss several aspects of the complexity of focus formation and present a model for the involvement of individual and complex recruitment mechanisms in focus formation.     

BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1

BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1-BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1-BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.     

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.     

BLM is an early responder to DNA double-strand breaks

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.