The MRN complex in double-strand break repair and telomere maintenance

Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end-joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here, we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

53BP1 promotes ATM activity through direct interactions with the MRN complex

The Mre11/Rad50/Nbs1 (MRN) complex has a central function in facilitating activation of the ATM protein kinase at sites of DNA double‐strand breaks (DSBs). However, several other factors are also required in human cells for efficient signalling through MRN and ATM, including the tumour suppressor proteins p53‐binding protein 1 (53BP1) and BRCA1. In this study, we investigate the functions of these mediator proteins in ATM activation and find that the presence of 53BP1 and BRCA1 can amplify the effects of MRN when interactions between MRN and ATM are compromised. This effect is dependent on a direct interaction between MRN and the tandem breast cancer carboxy‐terminal (BRCT) repeats in 53BP1, and is accompanied by hyper‐phosphorylation of both Nbs1 and 53BP1. We also find that the BRCT domains of 53BP1 affect the overall structure of 53BP1 multimers and that this structure is important for promoting ATM phosphorylation of substrates as well as for the repair of DNA DSBs in mammalian cells.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Five dysfunctional telomeres predict onset of senescence in human cells

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.     

The anaphase promoting complex promotes NHEJ repair through stabilizing Ku80 at DNA damage sites

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APC^Cdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.     

Budding yeast with human telomeres: a puzzling structure

Telomeres share some common features among eukaryotes, with few exceptions such as the fruit fly Drosophila that uses transposons as telomeres, they consist of G-rich repetitive DNA that is elongated by telomerase and/or alternative pathways depending on recombination. Telomere structure comprises both cis-acting satellite DNA (telomeric DNA) and proteins that interact directly and/or indirectly with the underlying DNA. Telomeric DNAs are surprisingly conserved among the vertebrates and very similar in most eukaryotes, but present some differences in yeast such as Saccharomyces cerevisiae. The telomeric proteins are more variable although the basic mechanisms which control telomere lengthening and capping are very similar, in fact orthologues of the yeast telomeric proteins, which have been studied first, have been identified in other organisms. Here we describe the structure of human telomeres in budding yeast as compared to canonical yeast and mammalian telomeres taking into consideration the more recent findings highlighting the mechanisms that are responsible for chromosome end protection and lengthening, and the role of chromatin organization in telomere function. This yeast represents a model for the study of mammalian telomeres that could be reconstituted step-by-step in all their components, moreover it could be useful for the assembly of mammalian artificial chromosome.     

MRN复合物的结构及功能研究进展

MRN复合物包括MRE11、NBS1、RAD50,此复合物中的MRE11或NBS1缺失或突变会导致人的共济失调一毛细血管扩张样疾病、Nijmegen断裂综合征。MRN复合物在DNA双链损伤修复、同源重组、非同源重组、端粒长度维持、细胞检验点激活、保证DNA复制的顺利进行,以及维持基因组的稳定性等方面都起到了重要的作用。从以上几个方面简要综述MRN复合物的研究进展。     

The function of classical and alternative non‐homologous end‐joining pathways in the fusion of dysfunctional telomeres

Repair of DNA double‐stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non‐homologous end‐joining (NHEJ) or error‐free homologous recombination. NHEJ precedes either by a classic, Lig4‐dependent process (C‐NHEJ) or an alternative, Lig4‐independent one (A‐NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere‐binding proteins are recognized as DSBs. In this report, we studied which end‐joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end‐to‐end chromosome fusions mediated by the C‐NHEJ pathway. In contrast, removal of Tpp1–Pot1a/b initiated robust chromosome fusions that are mediated by A‐NHEJ. C‐NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A‐NHEJ is a major pathway to process dysfunctional telomeres.     

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

The protein kinases ataxia‐telangiectasia mutated (ATM) and ATM‐Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild‐type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.     

The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeastSchizosaccharomyces pombe

We have recently demonstrated thatSchizosaccharomyces pombe cells treated with the nucleoside analogue 5-azacytidine (5-azaC) require previously characterised G2 checkpoint mechanisms for survival. Here we present a survey of known DNA repair mutations which defines those genes required for survival in the presence of 5-azaC. Using a combination of single-mutant and epistasis analyses we find that the excision, mismatch and recombinational repair pathways are all required in some degree for the repair of 5-azaC-mediated DNA damage. There are distinct differences in the epistatic interactions of several of the repair mutations with respect to 5-azaC-mediated DNA damage relative to UV-mediated DNA damage.     

A non‐canonical function of topoisomerase II in disentangling dysfunctional telomeres

The decatenation activity of topoisomerase II (Top2), which is widely conserved within the eukaryotic domain, is essential for chromosomal segregation in mitosis. It is less clear, however, whether Top2 performs the same function uniformly across the whole genome, and whether all its functions rely on decatenation. In the fission yeast, Schizosaccharomyces pombe, telomeres are bound by Taz1, which promotes smooth replication fork progression through the repetitive telomeric sequences. Hence, replication forks stall at taz1Δ telomeres. This leads to telomeric entanglements at low temperatures (⩽20°C) that cause chromosomal segregation defects and loss of viability. Here, we show that the appearance of entanglements, and the resulting cold sensitivity of taz1Δ cells, is suppressed by mutated alleles of Top2 that confer slower catalytic turnover. This suppression does not rely on the decatenation activity of Top2. Rather, the enhanced presence of reaction intermediates in which Top2 is clamped around DNA, promotes the removal of telomeric entanglements in vivo, independently of catalytic cycle completion. We propose a model for how the clamped enzyme–DNA complex promotes proper chromosomal segregation.     

Human RAP1 inhibits non‐homologous end joining at telomeres

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, promote genome stability by distinguishing chromosome termini from DNA double‐strand breaks (DSBs). Cells possess two principal pathways for DSB repair: homologous recombination and non‐homologous end joining (NHEJ). Several studies have implicated TRF2 in the protection of telomeres from NHEJ, but the underlying mechanism remains poorly understood. Here, we show that TRF2 inhibits NHEJ, in part, by recruiting human RAP1 to telomeres. Heterologous targeting of hRAP1 to telomeric DNA was sufficient to bypass the need for TRF2 in protecting telomeric DNA from NHEJ in vitro. On expanding these studies in cells, we find that recruitment of hRAP1 to telomeres prevents chromosome fusions caused by the loss of TRF2/hRAP1 from chromosome ends despite activation of a DNA damage response. These results provide the first evidence that hRAP1 inhibits NHEJ at mammalian telomeres and identify hRAP1 as a mediator of genome stability.     

A proteome-wide visual screen identifies fission yeast proteins localizing to DNA double-strand breaks

DNA double-strand breaks (DSBs) are a major threat to genome integrity. Proteins involved in DNA damage checkpoint signaling and DSB repair often relocalize and concentrate at DSBs. Here, we used an ORFeome library of the fission yeast Schizosaccharomyces pombe to systematically identify proteins targeted to DSBs. We found 51 proteins that, when expressed from a strong exogenous promoter on the ORFeome plasmids, were able to form a distinct nuclear focus at an HO endonuclease-induced DSB. The majority of these proteins have known connections to DNA damage response, but few have been visualized at a specific DSB before. Among the screen hits, 37 can be detected at DSBs when expressed from native promoters. We classified them according to the focus emergence timing of the endogenously tagged proteins. Eight of these 37 proteins are yet unnamed. We named these eight proteins DNA-break-localizing proteins (Dbls) and performed preliminary functional analysis on two of them, Dbl1 (SPCC2H8.05c) and Dbl2 (SPCC553.01c). We found that Dbl1 and Dbl2 contribute to the normal DSB targeting of checkpoint protein Rad26 (homolog of human ATRIP) and DNA repair helicase Fml1 (homolog of human FANCM), respectively. As the first proteome-wide inventory of DSB-localizing proteins, our screen result will be a useful resource for understanding the mechanisms of eukaryotic DSB response.     

SNMIB/Apollo protects leading‐strand telomeres against NHEJ‐mediated repair

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell''s inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo''s conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo^−/−ATM^−/− MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5′–3′ exonuclease function of SNM1B/Apollo in the generation of 3′ single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.     

Inhibition of peroxisome fission,but not mitochondrial fission,increases yeast chronological lifespan

Mitochondria are key players in aging and cell death. It has been suggested that mitochondrial fragmentation, mediated by the Dnm1/Fis1 organelle fission machinery, stimulates aging and cell death. This was based on the observation that Saccharomyces cerevisiae Δdnm1 and Δfis1 mutants show an enhanced lifespan and increased resistance to cell death inducers. However, the Dnm1/Fis1 fission machinery is also required for peroxisome division. Here we analyzed the significance of peroxisome fission in yeast chronological lifespan, using yeast strains in which fission of mitochondria was selectively blocked. Our data indicate that the lifespan extension caused by deletion of FIS1 is mainly due to a defect in peroxisome fission and not caused by a block in mitochondrial fragmentation. These observations are underlined by our observation that deletion of FIS1 does not lead to lifespan extension in yeast peroxisome deficient mutant cells.     

Tel1ATM dictates the replication timing of short yeast telomeres

Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double‐strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late‐replicating loci and that this effect on origin firing time is dependent on the Tel1^ATM checkpoint kinase. The effect of Tel1^ATM on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1^ATM specifies not only the extent but also the timing of telomerase recruitment.