Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

Abstract

The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such asrfa1-t11, are lethal in combination withrfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint geneRAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G₁/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G₂/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.

ACKNOWLEDGMENTS

We are grateful to Richard Kolodner for generously providingRFA1 mutants, to Christina DeCoste for assistance with FACS analysis, and to Vincent Geli, Suzanne Shanower, David Stern, Akio Sugino, and Ted Weinert for strains, plasmids, proteins, and antibodies. We also thank Nancy Walworth and laboratory members for helpful comments on the manuscript.

This work was supported by National Institutes of Health grant GM55583.