Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection.

Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5′-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs1-Dna2 and Exonuclease 1 (Exo1). Here we investigated the relationship between the three resection pathways with a focus on Exo1. Exo1 preferentially degrades the 5′-terminal stand of duplex DNA that is single stranded at the 3′ end, in agreement with its role downstream of the Mre11-Rad50-Xrs2 complex. Replication protein A (RPA) stimulates DNA end resection by Exo1 by both preventing nonspecific binding of Exo1 to and preventing degradation of single-stranded DNA. Nucleolytic degradation of DNA by Exo1 is inhibited by the helicase-deficient Sgs1 K706A mutant protein and, reciprocally, the nuclease-deficient Exo1 D173A mutant protein inhibits DNA unwinding by Sgs1. Thus, the activities of Sgs1 and Exo1 at DNA ends are mutually exclusive, establishing biochemically that both machineries function independently in DNA end processing. We also reconstituted Sgs1-Top3-Rmi1-RPA-Dna2 and Exo1 resection reactions both individually and combined, either with or without the Mre11-Rad50-Xrs2 complex. We show that the yeast Sgs1-Dna2 and Exo1 pathways do not stimulate one another and function as independent and separate DNA end-processing machineries, even in the presence of the stimulatory Mre11-Rad50-Xrs2 complex.