Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

Abstract

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Δ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Δ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

We thank N. Arnheim, S. Forsburg, and M. Goodman for sharing equipment; D. Arnold, W. Dolan, and J. Yuan for help with microscopy; J. Orr for excellent technical assistance; J. Bachant, F. Cross, S. Elledge, R. Rothstein, E. Schwob, S. Szyjka, D. Toczyski, and T. Weinert for strains and plasmids; M. Foiani for protocols; J. Bachant for critical reading of the manuscript; and S. Bell, J. Diffley, S. Elledge, and S. Haase for helpful discussions.

This work was supported by a Burroughs-Wellcome Career Award (992834) and NIH grant (1RO1GM-CA65494-01A1) to O.M.A.