BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.     

CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.     

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein. Despite the fact that RSF1 is often overexpressed in tumors and linked to tumorigenesis and genome instability, its role in the DSB response remains largely unclear. Here we show that RSF1 accumulates at DSB sites and protects human cells against IR-induced DSBs by promoting repair of these lesions through homologous recombination (HR) and non-homologous end-joining (NHEJ). Although SMARCA5 regulates the RNF168-dependent ubiquitin response that targets BRCA1 to DSBs, we found RSF1 to be dispensable for this process. Conversely, we found that RSF1 facilitates the assembly of centromere proteins CENP-S and CENP-X at sites of DNA damage, while SMARCA5 was not required for these events. Mechanistically, we uncovered that CENP-S and CENP-X, upon their incorporation by RSF1, promote assembly of the NHEJ factor XRCC4 at damaged chromatin. In contrast, CENP-S and CENP-X were dispensable for HR, suggesting that RSF1 regulates HR independently of these centromere proteins. Our findings reveal distinct functions of RSF1 in the 2 major pathways of DSB repair and explain how RSF1, through the loading of centromere proteins and XRCC4 at DSBs, promotes repair by non-homologous end-joining.     

Lif1 SUMOylation and its role in non-homologous end-joining

Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is inhibitory to Lif1 SUMOylation. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner.     

DNA-damage response and repair activities at uncapped telomeres depend on RNF8

Loss of telomere protection causes natural chromosome ends to become recognized by DNA-damage response and repair proteins. These events result in ligation of chromosome ends with dysfunctional telomeres, thereby causing chromosomal aberrations on cell division. The control of these potentially dangerous events at deprotected chromosome ends with their unique telomeric chromatin configuration is poorly understood. In particular, it is unknown to what extent bulky modification of telomeric chromatin is involved. Here we show that uncapped telomeres accumulate ubiquitylated histone H2A in a manner dependent on the E3 ligase RNF8. The ability of RNF8 to ubiquitylate telomeric chromatin is associated with its capacity to facilitate accumulation of both 53BP1 and phospho-ATM at uncapped telomeres and to promote non-homologous end-joining of deprotected chromosome ends. In line with the detrimental effect of RNF8 on uncapped telomeres, depletion of RNF8, as well as of the E3 ligase RNF168, reduces telomere-induced genome instability. This indicates that, besides suppressing tumorigenesis by mediating repair of DNA double-strand breaks, RNF8 and RNF168 might enhance cancer development by aggravating telomere-induced genome instability.     

Tel1 and Rif2 oppositely regulate telomere protection at uncapped telomeres in Saccharomyces cerevisiae

It has been well documented that Tel1 positively regulates telomere-end resection by promoting Mre11-Rad50-Xrs2(MRX) activity, while Rif2 negatively regulates telomere-end resection by inhibiting MRX activity. At uncapped telomeres, whether Tel1 or Rif2 plays any role remains largely unknown. In this work, we examined the roles of Tel1 and Rif2 at uncapped telomeres in yku70△ and/or cdc13-1 mutant cells cultured at non-permissive temperature. We found that deletion of TEL1 exacerbates the temperature sensitivity of both yku70△ and cdc13-1 cells. Further epistasis analysis indicated that MRX and Tel1 function in the same pathway in telomere protection. Consistently, TEL1 deletion increases accumulation of Exo1-dependent telomeric single-stranded DNA(ssDNA) at uncapped telomeres, which stimulates checkpoint-dependent cell cycle arrest. Moreover, TEL1 deletion in yku70△ cells facilitates Rad51-dependent Y0 recombination. In contrast, RIF2 deletion in yku70△ cells decreases the accumulation of telomeric ssDNA after 8 h of incubation at the non-permissive temperature of 37℃ and suppresses the temperature sensitivity of yku70△ cells, likely due to the increase of Mre11 association at telomeres.Collectively, our findings indicate that Tel1 and Rif2 regulate telomere protection at uncapped telomeres via their roles in balancing MRX activity in telomere resection.     

Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Delta telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1(AQ), a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Delta are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Delta strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.     

End-processing during non-homologous end-joining: a role for exonuclease 1

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5'-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.     

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.     

Mechanism and regulation of human non-homologous DNA end-joining

Non-homologous DNA end-joining (NHEJ)--the main pathway for repairing double-stranded DNA breaks--functions throughout the cell cycle to repair such lesions. Defects in NHEJ result in marked sensitivity to ionizing radiation and ablation of lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-receptor genes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic changes that underlie cancer and ageing.     

MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.     

BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.     

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.     

Antagonistic relationship of NuA4 with the non-homologous end-joining machinery at DNA damage sites

The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for Rad9 and NHEJ factors Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on Mre11-Rad50-Xrs2 (MRX) and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanisms in situations where some DNA-end resection can occur in G1.     

RPA provides checkpoint-independent cell cycle arrest and prevents recombination at uncapped telomeres of Saccharomyces cerevisiae

Replication Protein A (RPA) is an evolutionary conserved essential complex with single-stranded DNA binding properties that has been implicated in numerous DNA transactions. At damaged telomeres, Saccharomyces cerevisiae RPA recruits the Mec1–Ddc2 module of the DNA damage checkpoint network, its only known function in DNA damage signaling. Here, we describe rfa1 mutants (rfa1-1, rfa1-9, rfa1-10, rfa1-11 and rfa1-12) that are proficient in this checkpoint but nevertheless exhibit deregulation of cell cycle control upon telomere uncapping induced by the cdc13-1 mutation. Overriding of this damage-induced checkpoint-independent cell cycle block in the rfa1 mutants was suppressed following genetic inactivation of either TEL1 or EST2/telomerase. Altogether, our results suggest that a previously non-suspected function of RPA is to block cell cycle progression upon telomere uncapping using a yet unidentified pathway that functions in a Mec1–Ddc2-independent manner. We propose that in the rfa1 mutants, ill-masking of uncapped telomeres provokes inappropriate access of Tel1 and inappropriate functioning of telomerase, which, by yet unknown mechanisms, allows cell division to take place in spite of the block established by the DNA damage checkpoint. In the present study, we also observed that upon telomere uncapping, rfa1-12, but not the other studied rfa1 mutants, triggered telomeric recombination in the presence of functional telomerase. In conclusion, the present study identifies a novel pathway of telomere end protection that utilizes a previously unsuspected function of RPA at the telomeres.     

TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch

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New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining

The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all.     

The non-homologous end-joining protein Nej1p is a target of the DNA damage checkpoint

DNA double-strand breaks (DSBs), which are generated by ionizing radiation (IR) and a range of other DNA damaging agents, are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Previous studies have shown that NHEJ in Saccharomyces cerevisiae requires the Yku70p-Yku80p heterodimer and a complex consisting of DNA Ligase IV, Lif1p and Nej1p. Here, we report that Nej1p is phosphorylated in response to DNA damage in a manner that relies on the DNA damage checkpoint kinases Mec1p, Rad53p and Dun1p. By using a mutational approach, we have identified a consensus Dun1p phosphorylation site in Nej1p, and mutation of conserved serine residues within it leads to decreased NHEJ efficiency. These data, together with previous findings that Rad55p--a protein involved in HR--is phosphorylated analogously, point to there being a broad signalling network connecting DNA damage checkpoint responses with the regulation of DNA DSB repair activities.     

Cadmium inhibits non-homologous end-joining and over-activates the MRE11-dependent repair pathway

Although cadmium still represents a public health problem and despite the fact that it has been classified as an IARC Group-I carcinogen, the molecular and cellular mechanisms responsible for the toxicity and the carcinogenicity of cadmium compounds are poorly known. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key-lesions in cell lethality, and because misrepaired DSBs are a source of genomic instability leading to cancer proneness, the activity of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ) and recombination, has been evaluated in human endothelial cells exposed to cadmium chloride and cadmium diacetate. Exposure to cadmium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins, which was used as an indirect measure of DSB in this study. The presence of cadmium in cells decreases the repair rate of X-ray-induced DSBs, suggesting an impact of cadmium upon the reparability of DSBs. Such an interpretation was consolidated by the finding that the DNA-PK kinase activity, essential for NHEJ, is affected by the presence of cadmium. These results suggest that the toxicity of cadmium compounds may be explained by the propagation of persistent DSBs. In parallel, the presence of cadmium was also associated with an over-activation of the MRE11-dependent repair pathway that may favour genomic instability. Altogether, our data provide a first example of the impact of cadmium upon DSB repair and signalling.