A Role forSaccharomyces cerevisiaeChk1p in the Response to Replication Blocks

Abstract

, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1⌬ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1⌬ dun1⌬ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

ATRMec1 Phosphorylation-independent Activation of Chk1 in Vivo

“…To test this, we integrated CHK1L506R into dun1⌬ cells that also lacked Rad9. Rad9 functions as an adaptor protein for ATR Mec1 phosphorylation of Chk1 (5) and just like chk1⌬ dun1⌬ cells, dun1⌬ rad9⌬ cells die on low concentrations of HU (7). dun1⌬ rad9⌬ cells expressing wild-type Chk1 failed to grown on 50 mM HU; however, dun1⌬ rad9⌬ cells expressing Chk1L506R or Chk13AQL506R also lacking the ATR Mec1 phosphorylation sites grew as well as the wild-type cells on HU (Fig.…”

“…Although the Chk1/Rad9 DNA damage checkpoint arm is not required during replication blocks in the budding yeast, we have shown that Chk1 is activated when replication is slowed down by the ribonucleotide reductase inhibitor hydroxyurea (HU). 3 We have also shown that cells lacking the checkpoint kinase Dun1 are dependent on the Chk1/Rad9 arm for survival of replication blocks elicited by HU (7,8). Studies addressing the in vivo roles of mammalian Chk1 have been difficult due to the fact that Chk1 is essential for early development of mouse embryos (9).…”

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

“…Chk1 is part of a less well-characterized branch of the checkpoint response that lies parallel to Rad53 and Dun1 (Sanchez et al, 1999). Although Chk1 plays a crucial role in response to replication stress in fission yeast and in human cells, in budding yeast its role is only revealed when the Rad53-Dun1 branch is disabled; thus, chk1⌬ dun1⌬ strains are exceptionally sensitive to HU (Schollaert et al, 2004). As the synthetic HU sensitivity of the chk1⌬ dun1⌬ strain is similar to that of the ccr4⌬ dun1⌬ strain, we generated ccr4⌬ chk1⌬, chk1⌬ dun1⌬ and ccr4⌬ chk1⌬ dun1⌬ mutant strains to assess whether CHK1 lies in the same genetic pathway as CCR4.…”

“…Downstream of Mec1 and parallel to Rad53 and Dun1 lies the checkpoint kinase Chk1, which phosphorylates and stabilizes the anaphase inhibitor Pds1, also known as securin, to inhibit the G2-M transition (Wang et al, 2001). Chk1 plays an unidentified role in the response to replication stress, particularly in cells lacking DUN1 (Sanchez et al, 1999;Schollaert et al, 2004).…”

Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2

“…Mec1-dependent activation of the replication checkpoint also requires the adaptor Mrc1, the Saccharomyces cerevisiae homolog of human Claspin that forms a complex to stabilize replication forks at sites of replication stress (1,16,22,27). Additionally, although Chk1 is activated by Mec1 to promote anaphase arrest following DNA damage, it has also been proposed to play a role in response to hydroxyurea (HU)-induced replication blocks (8,32,34).…”