The Mechanism of Mus81-Mms4 Cleavage Site Selection Distinguishes It from the Homologous Endonuclease Rad1-Rad10

Abstract

ABSTRACT Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3′ branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae . We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5′ of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5′ end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5′ end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3′ flap recombination intermediate downstream of replication fork collapse.