Rfc5, a Replication Factor C Component, Is Required for Regulation of Rad53 Protein Kinase in the Yeast Checkpoint Pathway

Abstract

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae. We have previously shown that a temperature-sensitive (ts) rfc5-1 mutation is impaired in the S-phase checkpoint. In this report, we show that the rfc5-1 mutation is sensitive to DNA-damaging agents. RFC5 is necessary for slowing the S-phase progression in response to DNA damage. The phosphorylation of the essential central transducer, Rad53 protein kinase, is reduced in response to DNA damage in rfc5-1 mutants during the S phase. Furthermore, the inducibility of RNR3 transcription in response to DNA damage is dependent on RFC5. It has been shown that phosphorylation of Rad53 is controlled by Mec1 and Tel1, members of the subfamily of ataxiatelangiectasia mutated (ATM) kinases. We also demonstrate that overexpression of TEL1 suppresses the ts growth defect and DNA damage sensitivity of rfc5-1 mutants and restores phosphorylation of Rad53 and RNR3 induction in response to DNA damage in rfc5-1. Our results, together with the observation that overexpression of RAD53 suppresses the defects of the rfc5-1 mutation, suggest that Rfc5 is part of a mechanism transducing the DNA damage signal to the activation of the central transducer Rad53.In eukaryotic cells, successful mitotic division requires the events of the cell cycle to be ordered into dependent pathways in which the initiation of late cycle events is dependent on the completion of early events. The mechanisms which ensure that cell division does not occur before completion of such prerequisite steps have been termed checkpoint controls (8). Checkpoint controls ensure that cells remain in S phase before completion of DNA replication. DNA damage also activates checkpoint controls to provide enough time to complete DNA repair. Defects of these DNA-related checkpoints result in increased genomic instability and mutagenesis (5, 21).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 or G 2 /M and retard Sphase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase (5, 21). A number of genes that are involved in the DNA damage checkpoint and/or the replication checkpoint have been identified (5, 21). These include RAD9, RAD17, RAD24, POL2, MEC1/ESR1, RAD53/SPK1/MEC2/ SAD1, and MEC3 (1,10,17,28,[33][34][35]. Among these genes, RAD9, RAD17, RAD24, and MEC3 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (12,20,(25)(26)(27)(33)(34)(35). POL2, which encodes a large subunit of DNA polymerase ε (pol ε), is proposed to sense DNA damage and replication block in S phase (16,17). MEC1 and RAD53 are necessary for checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 35). RAD53 encodes a dual-specificity protein kinase (28), and Mec1 belongs to the phosphatidylinositol kinase family that includes S. cerevisiae Tel1 and human ATM proteins (10,15,24). MEC1 and TEL1 share some...

Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase ε in DNA Replication and the S/M Checkpoint Pathway

“…pol2-11 and pol2-12 mutants with defects in the S phase checkpoint were also defective in recognizing MMS-induced DNA damage. rfc5-1 mutants, defective in the DNA polymerase clamp loader, were also shown to have reduced damage-induced transcription and a defective S/M checkpoint (11,31). In the current study, we show that pol2-F appears to share the same behavior.…”

“…In fact, in only one case have a replication defect and a checkpoint defect been (partially) genetically separated. The overexpression of the POL30 gene, encoding the proliferating cell nuclear antigen was shown to suppress the replication defect of the rfc5 but not its checkpoint defect (11).…”

The Dna Replication Fork in Eukaryotic Cells

“…These defects were suppressed by mutations in the gene encoding PCNA (pol 30), supporting the biochemical interaction between these two proteins (128). Interestingly, a mutation in the RFC5 gene encoding the S. cerevisiae p38 subunit homolog causes a defect in a DNA-damage checkpoint signal that transmits to the Rad53 protein and the Tel1 protein, a yeast protein similar to the ataxia telangiectasia gene product ATM (129). This observation suggests that RFC might function in monitoring DNA damage at the replication fork.…”

“…The domain in pol ε that is responsible for its checkpoint function is separable from its DNA polymerase catalytic domain (272). Recent studies suggest that the Rad53p protein kinase is required for DNA-damage checkpoint signaling by a pol ε-and Rfc5p-dependent mechanism (129,273,275).…”

Colocalization of human Rad17 and PCNA in late S phase of the cell cycle upon replication block

“…In yeast, RF-C subunits also function in the DNA replication and DNA damage checkpoint control pathways. Fission and budding yeast cells carrying a deletion or mutation in genes encoding small RF-C subunits are defective in the DNA replication and DNA damage checkpoint functions (Reynolds et al, 1999;Shimada et al, 1999;Noskov et al, 1998;Gary Schmidt et al, 2001;Sugimoto et al, 1996Sugimoto et al, , 1997). An alternative RF-C complex consisting of the four small RF-C subunits and cell cycle checkpoint protein Rad24/Rad17 was isolated from uncompromised budding yeast (Green et al, 2000) and human cells (Lindsey-Boltz et al, 2001).…”