title: RAD51AP1-deficiency in vertebrate cells impairs DNA replication.
abstract: RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
authors.forename: Ann C
authors.initials: AC
authors.lastname: Parplys
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
authors.forename: Katja
authors.initials: K
authors.lastname: Kratz
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Speed
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
authors.forename: Stanley G
authors.initials: SG
authors.lastname: Leung
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States. Electronic address: dschild@lbl.gov.
authors.forename: David
authors.initials: D
authors.lastname: Schild
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States. Electronic address: claudia.wiese@colostate.edu.
authors.forename: Claudia
authors.initials: C
authors.lastname: Wiese
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line / Q000187:drug effects / Q000528:radiation effects; D002645:Chickens; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D011839:Radiation, Ionizing; D014714:Vertebrates / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C110410:RAD51AP1 protein, human; D016601:RNA-Binding Proteins; D051135:Rad51 Recombinase; D002945:Cisplatin; D006918:Hydroxyurea
keywords: DNA replication; DT40 cells; Homologous recombination; RAD51 foci; RAD51AP1