Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleasesin vivo

Abstract

Resection of the 5'-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods commonly used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with next-generation sequencing in which unique molecular identifiers allow exact counting of cleaved 5' ends at base pair resolution. Mutant strains, including exo1Δ, mre11-H125N, exo1Δ and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked an apparent motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB and near the Exo1 pause point and where it was much less restrained. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

“…In addition to its function in NHEJ, the MRX MRN complex is key to shifting repair towards HR when stimulated to initiate resection by Sae2. Indeed, Sae2 activates the endonuclease activity of MRX MRN , which cleaves the 5' strand of the DSB end (Bazzano et al, 2021;Cannavo and Cejka, 2014). This provides an entry point for MRX 3'-5' exonuclease activity, which degrades the DNA towards the DSB, creating a short ssDNA extensions that can no longer be ligated by the canonical NHEJ machinery (Cannavo and Cejka, 2014;Garcia et al, 2011;Mimitou and Symington, 2008).…”

Sir3 heterochromatin protein promotes non‐homologous end joining by direct inhibition of Sae2

“…In addition to its function in NHEJ, the MRX MRN complex is key to shifting repair towards HR when stimulated to initiate resection by Sae2. Indeed, Sae2 activates the endonuclease activity of MRX MRN , which cleaves the 5 0 strand of the DSB end (Cannavo & Cejka, 2014;Bazzano et al, 2021). This provides an entry point for MRX 3 0 -5 0 exonuclease activity, which degrades the DNA towards the DSB, creating short ssDNA extensions that can no longer be ligated by the canonical NHEJ machinery (Mimitou & Symington, 2008;Garcia et al, 2011;Cannavo & Cejka, 2014).…”