Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.
The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.
Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 2FA, UK.
Giacomo
G
De Piccoli
Yuki
Y
Katou
Takehiko
T
Itoh
Ryuichiro
R
Nakato
Katsuhiko
K
Shirahige
Karim
K
Labib
D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000502:physiology*; D042822:Genomic Instability; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D059807:S Phase Cell Cycle Checkpoints; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D013312:Stress, Physiological
D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae