Rfc5, in Cooperation with Rad24, Controls DNA Damage Checkpoints throughout the Cell Cycle in Saccharomyces cerevisiae

Abstract

RAD24 and RFC5 are required for DNA damage checkpoint control in thebudding yeast Saccharomyces cerevisiae. Rad24 is structurally relatedto replication factor C (RFC) subunits and associates with RFC subunitsRfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in allthe G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas therfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint.Both the RFC subunits and Rad24 contain a consensus sequence fornucleoside triphosphate (NTP) binding. To determine whether the NTP-bindingmotif is important for Rad24 function, we mutated the conserved lysine(115)residue in this motif. The rad24-K115E mutation, which changes lysineto glutamate, confers a complete loss-of-function phenotype, whilethe rad24-K115R mutation, which changes lysine to arginine, showsno apparent phenotype. Although neither rfc5-1 nor rad24-K115R singlemutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints,rfc5-1 rad24-K115R double mutants become defective in these checkpoints.Coimmunoprecipitation experiments revealed that Rad24(K115R) failsto interact with the RFC proteins in rfc5-1 mutants. Together, theseresults indicate that RFC5, like RAD24, functions in all the G(1)-,S- and G(2)/M-phase DNA damage checkpoints and suggest that the interactionof Rad24 with the RFC proteins is essential for DNA damage checkpointcontrol.