title: ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase.
abstract: The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.
authors.affiliation: University of Geneva, Department of Molecular Biology, Switzerland.
authors.forename: Kenji
authors.initials: K
authors.lastname: Shimada
authors.forename: Philippe
authors.initials: P
authors.lastname: Pasero
authors.forename: Susan M
authors.initials: SM
authors.lastname: Gasser
mesh_terms: D018797:Cell Cycle Proteins; D002470:Cell Survival / Q000187:drug effects; D064447:Checkpoint Kinase 2; D015825:Chromosomes, Fungal; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D018816:Genes, cdc / Q000502:physiology*; D006918:Hydroxyurea / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009154:Mutation; D009690:Nucleic Acid Conformation; D051738:Origin Recognition Complex; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018741:Replication Origin; D016196:S Phase / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497215:ORC2 protein, S cerevisiae; D051738:Origin Recognition Complex; D011500:Protein Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D003513:Cycloheximide; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea