Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes.
The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status.
Roslin Institute, Roslin, Midlothian, Scotland EH25 9PS, UK.
Juan
J
Domínguez-Bendala
Helen
H
Priddle
Alan
A
Clarke
Jim
J
McWhir
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation; D005786:Gene Expression Regulation / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use; D051379:Mice; D051135:Rad51 Recombinase; D011995:Recombination, Genetic / Q000235:genetics; D039901:Totipotent Stem Cells / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse