Abstract
Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Androstadienes / pharmacology
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Animals
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Ataxia Telangiectasia Mutated Proteins
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CDC2-CDC28 Kinases*
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Caffeine / pharmacology
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Carrier Proteins / metabolism
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Cell Cycle Proteins
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Cell-Free System
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Chromosomes / genetics
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Cyclin E / metabolism
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism
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DNA / genetics
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DNA Damage / physiology*
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DNA Replication / drug effects
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DNA Replication / physiology*
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DNA-Binding Proteins*
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Enzyme Inhibitors / pharmacology
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Male
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Mutagenesis / physiology
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Nuclear Proteins / metabolism
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Oocytes
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Phosphodiesterase Inhibitors / pharmacology
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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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Replication Origin / physiology
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S Phase / genetics
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Saccharomyces cerevisiae Proteins*
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Signal Transduction / physiology*
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Spermatozoa
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Transcription, Genetic / genetics
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Tumor Suppressor Proteins
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Tyrosine / metabolism
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Wortmannin
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Xenopus
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Xenopus Proteins
Substances
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Androstadienes
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CDC45 protein, S cerevisiae
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Carrier Proteins
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Cell Cycle Proteins
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Cyclin E
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DNA-Binding Proteins
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Enzyme Inhibitors
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Nuclear Proteins
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Phosphodiesterase Inhibitors
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Saccharomyces cerevisiae Proteins
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Tumor Suppressor Proteins
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Xenopus Proteins
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Caffeine
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Tyrosine
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DNA
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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Cdk2 protein, Xenopus
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases
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Wortmannin