Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 from Saccharomyces cerevisiae is a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.