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J. Biol. Chem., Vol. 276, Issue 38, 35458-35464, September 21, 2001
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From the University of Texas Health Science Center and Institute of
Biotechnology, San Antonio, Texas 78245
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.
DNA Structure-specific Nuclease Activities in the
Saccharomyces cerevisiae Rad50·Mre11 Complex*
*
This work was supported by National Institutes of Health
Research Grants R01 ES07061 and R01 GM57814.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Texas
Health Science Center and Institute of Biotechnology, 15355 Lambda Dr.,
San Antonio, TX 78245. E-mail: sung@uthscsa.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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