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J. Biol. Chem., Vol. 275, Issue 22, 16518-16529, June 2, 2000
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From the Braun Laboratory, California Institute of Technology,
Pasadena, California 91125
Saccharomyces cerevisiae Dna2 protein
is required for DNA replication and repair and is associated with
multiple biochemical activities: DNA-dependent ATPase, DNA
helicase, and DNA nuclease. To investigate which of these activities is
important for the cellular functions of Dna2, we have identified
separation of function mutations that selectively inactivate the
helicase or nuclease. We describe the effect of six such mutations on
ATPase, helicase, and nuclease after purification of the mutant
proteins from yeast or baculovirus-infected insect cells. A mutation in
the Walker A box in the C-terminal third of the protein affects
helicase and ATPase but not nuclease; a mutation in the N-terminal
domain (amino acid 504) affects ATPase, helicase, and nuclease. Two
mutations in the N-terminal domain abolish nuclease but do not reduce
helicase activity (amino acids 657 and 675) and identify the putative
nuclease active site. Two mutations immediately adjacent to the
proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity
in the presence of ATP. These results suggest that, although the Dna2
helicase and nuclease activities can be independently affected by some
mutations, the two activities appear to interact, and the nuclease
activity is regulated in a complex manner by ATP. Physiological
analysis shows that both ATPase and nuclease are important for the
essential function of DNA2 in DNA replication and for its
role in double-strand break repair. Four of the nuclease mutants are
not only loss of function mutations but also exhibit a dominant
negative phenotype.
The Nuclease Activity of the Yeast Dna2 Protein, Which Is Related
to the RecB-like Nucleases, Is Essential in
Vivo*
*
This work was supported by United States Public Health
Service Grants GM25508 and NSF 9507352.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: Braun Laboratory
147-75, California Institute of Technology, Pasadena, CA 91125. Tel.:
626-395-6053; Fax: 626-405-9452; E-mail:
jcampbel@cco.caltech.edu.
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