DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1.
To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.
Cobb
Jennifer A
JA
University of Geneva, Department of Molecular Biology, Quai Ernest-Ansermet 30, CH-1211 Geneva, Switzerland.
Bjergbaek
Lotte
L
Shimada
Kenji
K
Frei
Christian
C
Gasser
Susan M
SM
D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004271:DNA, Fungal / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008954:Models, Biological; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D018741:Replication Origin; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004259:DNA-Directed DNA Polymerase; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases; D006918:Hydroxyurea