title: 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 clb5Delta 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 clb5Delta cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Delta 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 clb5Delta cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.
authors.affiliation: Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1340, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gibson
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: Aparicio
authors.forename: Fangfang
authors.initials: F
authors.lastname: Hu
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Aparicio
mesh_terms: D016203:CDC2 Protein Kinase / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics; D002470:Cell Survival; D064447:Checkpoint Kinase 2; D019926:Cyclin B / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004789:Enzyme Activation; D017353:Gene Deletion; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013696:Temperature; D013997:Time Factors
chemical_list: C076558:CLB5 protein, S cerevisiae; C095227:CLB6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; C441579:MRC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase