Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae

The large subunit of replication protein A (Rpa1) consists of three single- stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown fu nction. To determine the essential role of this domain we searched for muta tions that require wild-type Rpa1N for viability in yeast. A mutation in RF C4, encoding a small subunit of replication factor C (RFC), was found to di splay allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synt hesis inhibitor hydroxyurea, such as rfal-t11, are lethal in combination wi th rfc4-2, The rfc4-2 mutant itself is sensitive to hydroxyurea, and like r fc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints, RFC4 and the DNA damage checkpoint gene RAD24 were fou nd to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G,IS DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA d amage checkpoint. Thus, in addition to its essential role as part of the cl amp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DN A checkpoint pathways. Our results suggest that a physical interaction betw een Rfc l and Rpa1N is required for both roles.