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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle Proteins*
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Cell Survival / drug effects
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Checkpoint Kinase 2
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Chromosomes, Fungal
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Cycloheximide / pharmacology
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DNA Damage
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DNA Replication
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DNA, Fungal / biosynthesis*
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DNA-Binding Proteins / physiology*
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Dose-Response Relationship, Drug
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Fungal Proteins / genetics
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Fungal Proteins / metabolism*
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Gene Expression Regulation, Fungal
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Genes, cdc / physiology*
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Hydroxyurea / pharmacology
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Methyl Methanesulfonate / pharmacology
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Mutation
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Nucleic Acid Conformation
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Origin Recognition Complex
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Phosphorylation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Synthesis Inhibitors / pharmacology
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Replication Origin
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S Phase / physiology*
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Saccharomyces cerevisiae / physiology*
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Saccharomyces cerevisiae Proteins*
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Ubiquitin / metabolism
Substances
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Cell Cycle Proteins
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DNA, Fungal
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DNA-Binding Proteins
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Fungal Proteins
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ORC2 protein, S cerevisiae
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Origin Recognition Complex
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Protein Synthesis Inhibitors
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Saccharomyces cerevisiae Proteins
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Ubiquitin
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Cycloheximide
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Methyl Methanesulfonate
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Checkpoint Kinase 2
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Protein Serine-Threonine Kinases
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RAD53 protein, S cerevisiae
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Hydroxyurea