Abstract
In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B, Member 3
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Animals
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Blotting, Western
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Chromatin / metabolism
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Chromatin Immunoprecipitation
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Comet Assay
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DNA Damage
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DNA Helicases / genetics
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DNA Helicases / metabolism
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DNA Replication*
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DNA, Single-Stranded
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Electrophoresis, Agar Gel
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Female
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MRE11 Homologue Protein
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Male
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Minichromosome Maintenance Complex Component 2
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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Mutation
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Oocytes / metabolism
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Proliferating Cell Nuclear Antigen / genetics
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Proliferating Cell Nuclear Antigen / metabolism
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Protein Binding
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Rad51 Recombinase / genetics
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Rad51 Recombinase / metabolism*
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Recombinant Proteins / metabolism
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Xenopus Proteins / genetics
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Xenopus Proteins / metabolism*
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Xenopus laevis
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 3
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Carrier Proteins
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Cdc45 protein, Xenopus
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Cell Cycle Proteins
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Chromatin
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DNA, Single-Stranded
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DNA-Binding Proteins
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Mre11 protein, Xenopus
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Multiprotein Complexes
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Proliferating Cell Nuclear Antigen
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Recombinant Proteins
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TAP2 protein, Xenopus
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Xenopus Proteins
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RAD51 protein, Xenopus
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Rad51 Recombinase
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MRE11 Homologue Protein
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DNA Helicases
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Mcm2 protein, Xenopus
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Minichromosome Maintenance Complex Component 2