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Papers In Press, published online ahead of print June 29, 2001
J. Biol. Chem, 10.1074/jbc.M011671200
Submitted on December 26, 2000
Revised on June 27, 2001
Accepted on June 28, 2001
Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110
Corresponding Author: burgers{at}biochem.wustl.edu
Replication Factor C is required to load PCNA onto primer-template junctions, using the energy of ATP hydrolysis. Four out 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.
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