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J. Biol. Chem., Vol. 276, Issue 37, 34792-34800, September 14, 2001
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From the Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine,
St. Louis, Missouri 63110
Replication factor C is required to load
proliferating cell nuclear antigen onto primer-template junctions,
using the energy of ATP hydrolysis. Four of the five RFC
genes have consensus ATP-binding motifs. To determine the relative
importance of these sites for proper DNA metabolism in the cell, the
conserved lysine in the Walker A motif of RFC1,
RFC2, RFC3, or RFC4 was mutated to
either arginine or glutamic acid. Arginine mutations in all
RFC genes tested permitted cell growth, although poor
growth was observed for rfc2-K71R. A glutamic acid
substitution resulted in lethality in RFC2 and
RFC3 but not in RFC1 or RFC4. Most
double mutants combining mutations in two RFC genes were
inviable. Except for the rfc1-K359R and
rfc4-K55E mutants, which were phenotypically similar to
wild type in every assay, the mutants were sensitive to DNA-damaging
agents. The rfc2-K71R and rfc4-K55R mutants
show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was
impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No
dramatic defects in telomere length regulation were detected in the
mutants. These data demonstrate that the ATP binding function of
RFC2 is important for both DNA replication and checkpoint
function and, for the first time, that RFC4 also plays a
role in checkpoint regulation.
ATP Utilization by Yeast Replication Factor C
IV. RFC ATP-BINDING MUTANTS SHOW DEFECTS IN DNA REPLICATION, DNA
REPAIR, AND CHECKPOINT REGULATION*,
*
This work was supported in part by Grant GM32431 from the
National Institutes of Health.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.
The on-line version of this article (available at
http://www.jbc.org) contains figures for the Appendix.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biophysics, Washington University
School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: burgers@biochem.wustl.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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