The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress.

Naomi Coulton,Thomas Caspari

doi:10.1242/bio.029272

Abstract

ABSTRACTWhile mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10) and is epistatic with a mutation in DNA polymerase delta (cdc6.23) when DNA is alkylated by methyl-methanesulfate (MMS). The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50) is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

Highlights

Schizosaccharomyces pombe Chk1 is phosphorylated in its Cterminal domain at serine-345 by Rad3 (ATR) after the kinase is recruited to a broken chromosome by Rad4 (TopBP1), Crb2 (53BP1) and the Rad9-Rad1-Hus1 ring (Capasso et al, 2002; Lopez-Girona et al, 2001; Saka et al, 1997; Furuya et al, 2004)
The only separation-of-function conditions known so far are the phosphorylation of S317 of human Chk1, which is only required for the DNA damage response but not for its essential functions (Wilsker et al, 2008), and the mutations E92D and I484T in S. pombe Chk1 which affect the S-M checkpoint but only at 37°C (Francesconi et al, 1997)
We report here a new separation-offunction mutation, S173 to alanine (S173A), in the kinase domain of S. pombe Chk1, that abolishes the G1-M arrest in a cdc10.V50 strain background and the S-M checkpoint in HU-treated Δcds1 cells without affecting the G2-M arrest in the presence of exogenous DNA damaging agents

Summary

Introduction

Schizosaccharomyces pombe Chk is phosphorylated in its Cterminal domain at serine-345 by Rad (ATR) after the kinase is recruited to a broken chromosome by Rad (TopBP1), Crb (53BP1) and the Rad9-Rad1-Hus ring (Capasso et al, 2002; Lopez-Girona et al, 2001; Saka et al, 1997; Furuya et al, 2004). Activated Chk delays cell-cycle progression at the G2-M boundary by stimulating Wee to phosphorylate the inhibitory tyrosine-15 residue of Cdc (CDK1) kinase and by simultaneously removing the activating tyrosine phosphatase Cdc from the nucleus (Furnari et al, 1999; O’Connell et al, 1997). Received August 2017; Accepted October 2017 performs a second, more enigmatic role in G1 where it prevents premature mitosis when the transcription factor Cdc is impaired (Carr et al, 1995). It phosphorylates Cdc in the presence of methyl-methanesulfonate (MMS) that alkylates the DNA template to delay G1-S transition (Ivanova et al, 2013)

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