title: RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.
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.
authors.affiliation: Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK.
authors.forename: Yoshitami
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Fabio
authors.initials: F
authors.lastname: Puddu
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Costanzo
mesh_terms: D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D020552:Comet Assay; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005260:Female; D000076228:MRE11 Homologue Protein; D008297:Male; D064111:Minichromosome Maintenance Complex Component 2; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D002352:Carrier Proteins; C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C510917:Mre11 protein, Xenopus; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; C489515:TAP2 protein, Xenopus; D029867:Xenopus Proteins; C495152:RAD51 protein, Xenopus; D051135:Rad51 Recombinase; D000076228:MRE11 Homologue Protein; D004265:DNA Helicases; C102186:Mcm2 protein, Xenopus; D064111:Minichromosome Maintenance Complex Component 2