title: RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells.
abstract: The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.
authors.affiliation: The Institute for Cancer Studies, University of Sheffield, Sheffield, UK.
authors.forename: Kate M
authors.initials: KM
authors.lastname: Sleeth
authors.forename: Claus Storgaard
authors.initials: CS
authors.lastname: Sørensen
authors.forename: Natalia
authors.initials: N
authors.lastname: Issaeva
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Dziegielewski
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Thomas
authors.initials: T
authors.lastname: Helleday
mesh_terms: D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004789:Enzyme Activation; D018816:Genes, cdc; D006801:Humans; D006918:Hydroxyurea / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D006918:Hydroxyurea