Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance.
The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.
Bjergbaek
Lotte
L
Department of Molecular Biology and NCCR, Frontiers in Genetics, University of Geneva, Quai Ernest-Ansermet 30, Geneva, Switzerland.
Cobb
Jennifer A
JA
Tsai-Pflugfelder
Monica
M
Gasser
Susan M
SM
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000502:physiology; D064447:Checkpoint Kinase 2; D004265:DNA Helicases / Q000502:physiology*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000502:physiology; D006918:Hydroxyurea / Q000494:pharmacology; D047468:Immunoprecipitation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D053484:RecQ Helicases; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I; D006918:Hydroxyurea