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 clb5⌬ 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 clb5⌬ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5⌬ 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 clb5⌬ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.Duplication of eukaryotic chromosomes involves the initiation of DNA synthesis from multiple origins of replication distributed along each chromosome. Although chromosomal DNA replication is restricted to the S-phase period of the cell cycle, individual replication origins initiate DNA synthesis (or "fire") at different times during S phase in a regulated fashion such that each replication origin has a characteristic initiation time within S phase. The exact nature of this regulation is not fully understood but appears to be influenced by two apparently unrelated factors: the local chromatin environment of each origin, which establishes the relative order of origin firing prior to S phase (reviewed in reference 43); and checkpoint signaling pathways, which modulate the extent to which the initiation of certain origins is delayed, particularly in response to replication defects or DNA damage (21,29,31,33).The pre-replicative complex (pre-RC) assembles at and governs the function of each replication origin (reviewed in reference 4). Activation of the pre-RC results in its conversion into two, divergent, replication fork complexes (replisomes) through the recruitment of additional DNA synthesis factors, such as Cdc45 and DNA polymerases. Pre-RC activation requires the function of cyclin-dependent kinase (CDK), which consists of a catalytic subunit (Cdk1) controlled by a cyclin subunit whose expression and stability are cell cycle regulated. In Saccharomyces ce...

Swe1 regulation and transcriptional control restrict the activity of mitotic cyclins toward replication proteins in Saccharomyces cerevisiae

“…2C). Together with recent data indicating that increased Clb6 dosage can drive late origin firing (22), these data demonstrate that early and late-firing replication origins can use any Clb-Cdk1 for activation (however, Clb1 remains untested).…”

“…Spore analysis has been described in ref. 22. Two-dimensional agarose gel electrophoresis and DNA content analyses have been described in ref.…”

A Dbf4p BRCA1 C-Terminal-Like Domain Required for the Response to Replication Fork Arrest in Budding Yeast

“…Therefore, we examined Rad53p abundance and activation in synchronous cultures of the WT, dbf4-DN109, dbf4-DN221, and clb5D strains passing through S-phase ( Figure 7B). We examined the clb5D strain as a control since it has a prolonged S-phase (Schwob and Nasmyth 1993) and is also synthetically lethal with rad53D sml1D, likely reflecting a requirement for activated Rad53p in late S-phase (Gibson et al 2004). Wild-type cells arrested with a-factor had low levels of Rad53p (Sanchez et al 1996).…”

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

“…Raffinose was present at 2% in YEP-Raffinose. Cell culturing, synchronization, DNA content analysis (FACScan), and Rad53 analysis have been described Gibson et al 2004), except we used anti-Rad53 antibody at 1:1000 (Santa Cruz Biotechnology, SC6749). The in situ Rad53 kinase assay is described in Pellicioli et al (1999).…”