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

Abstract

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 ...

Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP

“…We have been unable to detect a direct interaction between CtIP and DNA2 (data not shown), but it remains possible that they associate on DNA. Consistent with our model, cell-based assays have shown an epistatic relationship between these proteins (Hoa et al, 2015). We note that S. pombe Ctp1 has high affinity for the fork structure and also a DNA end bridging activity, suggesting a function of CtIP in creating a high localized concentration of DNA ends to accord nucleases and helicases easier access (Andres et al, 2015).…”

“…Moreover, recent findings suggest that Sae2 facilitates the removal of MRX from resected DSBs (Chen et al, 2015; Lisby et al, 2004; Puddu et al, 2015). Epistatic interactions between CtIP and Dna2 have been reported in chicken DT40 and human cells (Hoa et al, 2015), and depletion of CtIP and Dna2 confer comparable resection defects in Xenopus egg extracts (Peterson et al, 2013). …”

A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1

“…No dramatic reduction in the generation of cytosolic ssDNA was observed in Mre11-depleted cells. Mre11 is known to play an important role in resection (Cejka 2015), but its requirement in resection of IR-induced DSBs has been reported recently to be only partial in genomically disrupted human cells (Hoa et al 2015).…”

TDP1 is Critical for the Repair of DNA Breaks Induced by Sapacitabine, a Nucleoside also Targeting ATM- and BRCA-Deficient Tumors

“…Nucleotides indicated by capital letters in in F2 and R2 are identical with sequences upstream and downstream of the AflII site. Transfection was done as described previously (20). TDP1 KO clones were identified by genomic PCR using F3/R3 (for NEO R ) and F4/R3 (for PURO R ).…”