A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5-3 exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.Genetic and biochemical studies have indicated eukaryotes contain a mismatch repair (MMR) pathway related to the bacterial MutHLS pathway (reviewed in refs. 1 and 2). However, recent evidence suggests that eukaryotic MMR is more complex. In Saccharomyces cerevisiae there are two MMR pathways that require MSH2, a MutS homologue that recognizes mispaired bases (3, 4). One is a single base substitution mispair pathway that requires a complex of MSH2 and the MutS homologue MSH6 (also called GTBP or p160 in humans) (1-3). There is also an insertion͞deletion mispair pathway that requires either a complex of MSH2 and MSH6 or a complex of MSH2 and MSH3, a third MutS homologue (1-3). Additionally, four S. cerevisiae MutL homologues have been identified, PMS1 (PMS2 in humans) and MLH1-MLH3; PMS1 and MLH1 function in MMR and have been shown to form a heterodimer (1, 2).In vitro studies in Escherichia coli have shown that the excision step of MMR can occur either 5Ј to 3Ј or 3Ј to 5Ј of the initiating nick and requires the combination of a helicase (UvrD) and one of three single-stranded DNA exonucleases (Exo I, Exo VII or RecJ) (reviewed in ref. 2). In eukaryotic MMR, proteins involved in excising the mispair have not been identified, although some candidates have been suggested. These include S. cerevisiae RAD27 (RTH1, YKL510), a 5Ј-3Ј exonuclease and flap endonuclease (5), and Schizosaccharomyces pombe EXO1 and its Drosophila homologue Tosca, which are members of the same family of endo-and exonucleases as RAD27 (6, 7).The importance of determining the mechanism of MMR is underscored by its association with hereditary nonpolyposis colorectal carcinoma (HNPCC) (reviewed in refs. 2 and 8). HNPCC is associated primarily with germ-line mutations in two human MMR genes, MSH2 and MLH1, whereas mutations in other MMR genes are rare (ref. 9; reviewed in refs. 2 and 8). Somatic mutations in MMR genes have been found in some sporadic tumors, suggesting some sporadic cancers could be due to acquired mutations in MMR genes (reviewed in ref. 8, and see ref. 10). However, not all of HNPCC or sporadic cancers with mutator phenotypes can be accounted for by known MMR genes (9, 10). Consequently, there has been interest in identifying additional MMR genes. Here we describe the use of a two-hybrid screen to identify proteins that interact with MSH2 and function...

Using a substrate measuring deletion or inversion of an I-SceI-excised fragment and both accurate and inaccurate rejoining, we determined the impact of non-homologous end-joining (NHEJ) on mammalian chromosome rearrangements. Deletion is 2- to 8-fold more efficient than inversion, independent of the DNA ends structure. KU80 controls accurate rejoining, whereas in absence of KU mutagenic rejoining, particularly microhomology-mediated repair, occurs efficiently. In cells bearing both the NHEJ and a homologous recombination (HR) substrate containing a third I-SceI site, we show that NHEJ is at least 3.3-fold more efficient than HR, and translocation of the I-SceI fragment from the NHEJ substrate locus into the HR-I-SceI site can occur, but 50- to 100-fold less frequently than deletion. Deletions and translocations show both accurate and inaccurate rejoining, suggesting that they correspond to a mix of KU-dependent and KU-independent processes. Thus these processes should represent prominent pathways for DSB-induced genetic instability in mammalian cells.

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV–dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

Here we have used an intrachromosomal substrate to monitor the end joining of distant ends, which leads to DNA rearrangements in mammalian cells. We show that silencing Mre11 reduces the efficiency of nonhomologous end joining (NHEJ), affecting both the canonical and alternative pathways, partly in a manner that is independent of the ataxia-telangiectasia mutated kinase (ATM). Silencing of Rad50 or CtIP decreases end-joining efficiency in the same pathway as Mre11. In cells defective for Xrcc4, the MRE11-RAD50-NBS1 (MRN) complex inhibitor MIRIN decreases end-joining frequencies, demonstrating a role for MRN in alternative NHEJ. Consistently, MIRIN sensitizes both complemented and NHEJ-defective cells to ionizing radiation. Conversely, overexpression of Mre11 stimulates the resection of single-stranded DNA and increases alternative end joining, through a mechanism that requires Mre11's nuclease activity, but in an ATM-independent manner. These data demonstrate that, in addition to its role in ATM activation, Mre11 can favor alternative NHEJ through its nuclease activity.

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.

XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal doublestrand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

Germ-line mutations in the BRCA1 gene strongly predispose women to breast cancer (lifetime risk up to 80%). Furthermore, the BRCA1 protein is absent or present at very low levels in about one third of sporadic breast cancers. However, the mechanisms underlying BRCA1 somatic inactivation appear multiple and are still not fully understood. We report here the involvement of miR-146a and miR-146b-5p that bind to the same site in the 3′UTR of BRCA1 and down-regulate its expression as demonstrated using reporter assays. This was further confirmed with the endogenous BRCA1 gene by transfecting microRNA (miRNA) precursors or inhibitors in mammary cell lines. This down-regulation was accompanied by an increased proliferation and a reduced homologous recombination rate, two processes controlled by BRCA1. Furthermore, we showed that the highest levels of miR-146a and/or miR-146b-5p are found in basal-like mammary tumour epithelial cell lines and in triple negative breast tumours, which are the closest to tumours arising in carriers of BRCA1 mutations. This work provides further evidence for the involvement of miRNAs in sporadic breast cancer through down-regulation of BRCA1.

The tumor suppressor protein p53 controls cell cycle checkpoints and apoptosis via the transactivation of several genes. However, data from various laboratories suggest an additional role for p53: transcription-independent suppression of homologous recombination (HR). Genetic and physical interactions among p53, HR proteins (e.g. RAD51 and RAD54) and HR-DNA intermediates show that p53 acts directly on HR during the early and late steps of recombination. Complementary to the MSH2 mismatch-repair system, p53 appears to impair excess HR by controlling the minimal efficiency processing segment and by reversing recombination intermediates. By controlling the balance between the BLM and the RAD51 pathways, this direct role of p53 could maintain genome stability when replication forks are stalled at regions of DNA damage. In this article, we discuss the direct role of p53 on HR and the consequences for genome stability, tumor protection and speciation.