Mre11 Is Essential for the Removal of Lethal Topoisomerase 2 Covalent Cleavage Complexes.

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-/H129N) 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-/H129N 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-/H129N 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.

Relative contribution of four nucleases, CtIP, Dna2, Exo1 and Mre11, to the initial step of DNA double-strand break repair by homologous recombination in both the chicken DT40 and human TK6 cell lines.

Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' 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.

The role of the Mre11-Rad50-Nbs1 complex in double-strand break repair-facts and myths.

Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5'-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51-DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation-induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution.

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.