Coincident Resection at Both Ends of Random, γ–Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease

Abstract

Resection is an early step in homology-directed recombinational repair (HDRR) of DNA double-strand breaks (DSBs). Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced “dirty” DSBs or even enzyme-induced “clean” DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX) at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility (“PFGE-shift”). Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN) null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP) and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at “dirty” and clean DSB ends. These approaches apply to resection at other DSBs. Given evolutionary conservation, the observations are relevant to DNA repair in human cells.

Structural Studies of DNA End Detection and Resection in Homologous Recombination

“…Blocked DNA ends occur in meiosis or during abortive topoisomerase reactions Longhese et al 2009). The MRN(X) complex and Sae2/CtIP may indeed be dispensable for the resection of "clean" DNA ends, as shown for HO-endonuclease and I-SceI-induced DSBs in yeast (Llorente and Symington 2004;Westmoreland and Resnick 2013). In contrast to a model, in which MRN(X) and Sae2/CtIP start resection directly at a DNA end, newer studies provide an alternative model, in which MRN(X) incises DNA away from the DNA end (Fig.…”

Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

“…Unlike S1 nuclease, which also acts on ssDNA nicks (26,27), MBN is specific to ssDNA tails and gaps. Previously, we had addressed resection at random DSBs and found that duplex DNA with resected ssDNA tails of a few hundred to thousands of bases also exhibited retarded mobility on PFGE (referred to as "PFGE-shift") with a maximum apparent increase in molecular weight of 150 kb (18,28). However, there was no evidence of SMD or trapping of DNA in the well.…”

The global role for Cdc13 and Yku70 in preventing telomere resection across the genome

“…NotI restriction enzyme treatments of sample plugs in Supplementary Figs. S4E and S4F were described in [10]. All PFGE separations were performed with a CHEF Mapper XA system (Bio-Rad, Hercules, CA).…”

“…For the PFGE conditions used in this study, short resection tracks of ~200 bases or less are barely detectable [9], but increasing the length of single-stranded ends up to ~3 kb progressively increases the mobility shift to a maximum apparent increase in chromosome size of about 40 kb (for 1-end resections) and about 140 kb (for 2-end resections). Extensive resection lengths greater than ~3 kb do not cause further increase in the apparent size of resected chromosomes [8,10]. Importantly, this difference between the maximum PFGE-shift for 1-end vs 2-end resections enables the assay to distinguish between 1-end and 2-end extensive resection events.…”

“…Previously, we demonstrated that in a cdc13-1 mutant, the resulting resection could lead to PFGE-shift in some of the chromosomes in a population of cells [8]. However, we did not address 1- vs 2-end events as we had done for IR-induced DSBs [10]. Notably, we have subsequently developed conditions and identified genetic backgrounds in which yku70Δ cdc13-1 double mutants were not synthetically lethal, allowing assessment of the total contribution of each component to end resection of individual chromosomes as well as globally.…”