Abstract
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.
Highlights
Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis
There is a remarkable degree of agreement between the growth phenotype and the in vitro clamp loading defect, indicating that the major and essential function of Replication factor C (RFC) in the cell is that of clamp loading
This agreement is immediately apparent for the rfc2E and rfc3E mutations, which are lethal and result in vitro in an almost total defect in clamp loading
Summary
RFC ATP-BINDING MUTANTS SHOW DEFECTS IN DNA REPLICATION, DNA REPAIR, AND CHECKPOINT REGULATION*□S. Our biochemical studies reported in the second paper [4] of this series show that four ATP molecules can bind to RFC when PCNA and primer-template DNA are present This coincides with our current understanding of the primary sequence determinants of ATP-binding domains, which indicates that four of the five subunits comprising the RFC complex contain consensus ATP-binding motifs. The RFC-1E complex (containing Rfc1-K359E) was essentially like wild type, contrasting sharply with reports of similar mutations in human RFC, which abrogated the clamp loading activity of the mutant complex, and suggesting that ATP binding to Rfc is not essential for clamp loading in yeast (6 – 8). Mutations in other DNA replication genes, namely the catalytic subunit of DNA polymerase ⑀, POL2 [15] and a polymerase ⑀ associated gene DPB11 [16, 17], show defects in the S phase checkpoint
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call