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 the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Δ mutants are defective in all the G₁-, S-, and G₂/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine¹¹⁵ residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neitherrfc5-1 nor rad24-K115R single mutants are defective in the G₁- and G₂/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24^K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G₁-, S- and G₂/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.

ACKNOWLEDGMENTS

We thank A. Sugino for materials, M. Lamphier for critical readings of the manuscript, M. Mayer for discussion, and C. Green and N. Lowndes for communicating results prior to publication. K.S. acknowledges H. Mishima, Y. Miyake, H. Takahashi and H. Terasaki for suggestions and encouragement.

T.K. is a recipient of a JSPS predoctoral fellowship. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas and General Research from the Ministry of Education, Science, Sports and Culture of Japan (K.M. and K.S.).