Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires resection of 5′-termini to generate 3′-single-strand DNA tails1. Key components of this reaction are Exonuclease 1 and the bifunctional endo/exonuclease, Mre112-4. Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein—such as by the DNA end-joining complex5, topoisomerases6, or the meiotic nuclease, Spo117-13—but a specific function for the Mre11 3′-5′ exonuclease activity has remained elusive. Here, we reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5′-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated resection to traverse unidirectionally1. Using a combination of physical assays for 5′-end-processing, our results suggest an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5′-terminus of the DSB—much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5′-3′ direction away from the DSB, and Mre11 in the 3′-5′ direction towards the DSB end. Finally, Mre11 exonuclease activity confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways.

Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins

“…The results presented here, together with the literature data (22,42,45,49,51), point out the fact that ScMre11 possesses certain functional characteristics of ssDNA-binding protein. ScMre11has a 5-fold higher affinity for single-and doublestranded DNA as the length was increased from 40-to 70-mer ssDNA or 40-to 90-bp duplex DNA.…”

“…These intermediates are subjected to extensive resection by the concerted action either by Sgs1/Top3/RmiI, Dna2, and RPA or ExoI and RPA to generate long 3Ј ssDNA (which ranges from ϳ500 nucleotides to a few kilobases in size) (22,33,34,61,(82)(83)(84)(85)(86)(87)(88)(89). Recent studies have revealed that Sgs1, Dna2, and RPA constitute a minimal set of proteins needed for resection of DSBs in an ATP-dependent manner, and the MRX complex stimulates the end resection activity (61).…”

“…Furthermore, the human Mre11-Rad50-Nbs1 complex has been shown to possess weak ATP-stimulated DNA unwinding and does not need ends for DNA binding activity (49,50), and hMre11 by itself can bind both ssDNA and dsDNA and catalyze the annealing of complementary ssDNA molecules (51). Intriguingly, Mre11 nuclease activity is dispensable for the resection of DSBs in vegetatively growing yeast cells, although it is essential for the processing of meiotic DSBs, which are covalently bound on the 5Ј strand to the Spo11 protein (21,22,28,34,36,41). Further complicating the picture, Mre11 nuclease activities are not required for the activation of ATM after DNA damage in mammals (52).…”

“…Recent genetic data define a two-step mechanism for DSB end resection. First, the nuclease activity of MRX-Sae2 complex generates 50 -100 nucleotide 3Ј overhangs (1,4,22,35). The second step involves two pathways and two sets of components that promote bulk resection (1,4,22,35).…”

The Recombinases DMC1 and RAD51 Are Functionally and Spatially Separated during Meiosis in Arabidopsis

“…Work mostly performed in Saccharomyces cerevisiae and Schizosaccharomyces pombe demonstrated that the SPO11 proteins are subsequently released together with a short DNA oligonucleotide by an endonucleolytic cleavage adjacent to the break site, mediated by the Mre11/Rad50/Xrs2-Nbs1 (MRX/N) complex in conjunction with Sae2/Com1 (Neale et al, 2005;Hartung et al, 2007;Uanschou et al, 2007;Milman et al, 2009). The DNA break sites therefore have, as demonstrated for S. cerevisiae and S. pombe, protruding 39 termini, which are further extended by resection of the 59 termini depending on the activities of the MRX/N complex and Exo1 (Cromie and Smith, 2008;Mimitou and Symington, 2008;Farah et al, 2009;Manfrini et al, 2010;Zakharyevich et al, 2010;Garcia et al, 2011). The extensive single-stranded DNA (ssDNA) overhangs are bound by the heterotrimeric replication protein A (RPA) complex with high affinity, a prerequisite for the loading of the strand-exchange proteins Rad51 and Dmc1 (reviewed in Fanning et al, 2006;Broderick et al, 2010).…”