DNA structure-specific nuclease activities in the Saccharomyces cerevisiaeRad50 center dot Mre11 complex

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleol ytic processing of DNA double-strand breaks. We have overexpressed Rad50 an d Mre11 in yeast cells and purified them to near homogeneity. Consistent wi th the genetic data, we show that the purified Rad50 and Mre11 proteins for m a stable complex. In the Rad50(.)Mre11 complex, the protein components ex ist in equimolar amounts. Mre11 has a 3 ' to 5 ' exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not s ignificantly alter the exonucleolytic function of Mre11. Using homopolymeri c oligonucleotide-based substrates, we show that the exonuclease activity o f Mre11 and Rad50(.)Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radio-la beled hairpin structures that also contain 3 ' and 5 ' single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3 ' single-strande d DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculat e that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5 ' strand at secondary structures formed upon DNA strand separation.