The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11) and nuclease-deficient Mre11 (MRE11) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11 and MRE11 cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11 and MRE11 cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.

Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3 0 single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR. IntroductionDNA double-strand breaks (DSBs) are the most dangerous DNA damage, as a single unrepaired DSB can trigger apoptosis. DSBs are generated during physiological replication and induced by ionizingradiation. DSBs are repaired by two major DSB-repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice of DSB-repair pathway depends on the cell-cycle phase and the DNA-damaging agent (Symington & Gautier 2011). HR repairs DSBs in the S to G 2 phases, whereas NHEJ operates in all the cell phases. HR is more prominent than NHEJ in the repair of DSBs occurring during DNA replication (Hochegger et al. 2006;Qing et al. 2011) and is essential for cellular proliferation. Indeed, loss of critical HR factors, including CtIP, Mre11 and Rad51, causes mortality due to severe genome instability (Yamazoe et al. 2004;Nakamura et al. 2010;Hoa et al. 2015).HR is carried out in a series of steps, beginning with the 5 0 -to-3 0 strand resection of DSBs, which is called DSB resection (reviewed in Stracker & Petrini 2011;Symington & Gautier 2011). The resulting 3 0 -overhang is coated with a single-strand DNA binding protein, replication protein A (RPA). RPA is subsequently replaced with polymerized Rad51 recombinase, which polymerization results in the formation of subnuclear Rad51 foci. Polymerized Rad51 performs homology search and strand invasion into intact homologous sequences leading to formation of D-loop and Holliday junction structures. Biochemical and genetic studies have shown that in Saccharomyces cerevisiae (S. cerevisiae), DSB resection is initiated by Mre11 nuclease, which physically associates with Rad50 and Xrs2 (the MRX complex). The MRX complex and Sae2 are the orthologs of human Mre11/ Rad50/Nbs1 (the MRN complex) and CtIP, respectively. Yeast MRX ...

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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.

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