DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex

Abstract

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50.Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3' to 5' exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50.Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3' and 5' single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3' single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5' strand at secondary structures formed upon DNA strand separation.