title: An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks.
abstract: DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8.
authors: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
authors: Anna Maria
authors: AM
authors: Hegnauer
authors: Nicole
authors: N
authors: Hustedt
authors: Kenji
authors: K
authors: Shimada
authors: Brietta L
authors: BL
authors: Pike
authors: Markus
authors: M
authors: Vogel
authors: Philipp
authors: P
authors: Amsler
authors: Seth M
authors: SM
authors: Rubin
authors: Fred
authors: F
authors: van Leeuwen
authors: Aude
authors: A
authors: Guénolé
authors: Haico
authors: H
authors: van Attikum
authors: Nicolas H
authors: NH
authors: Thomä
authors: Susan M
authors: SM
authors: Gasser
mesh_terms: D000483:Alleles; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C502802:RPA70A protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004256:DNA Polymerase I; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases