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

Juan Dominguez-Bendala, Helen Priddle, Alan Clarke, Jim McWhir

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)298-307
Number of pages10
JournalExperimental Cell Research
Volume286
Issue number2
DOIs
StatePublished - Jun 10 2003

Fingerprint

Gene Targeting
Homologous Recombination
p53 Genes
Embryonic Stem Cells
Gene Frequency
Hypoxanthine Phosphoribosyltransferase
Bacterial Genes
Electroporation
Genetic Recombination
Down-Regulation
Clone Cells
DNA
Genes

Keywords

  • ES cells
  • Gene targeting
  • Homologous recombination
  • HPRT
  • p53
  • Rad51

ASJC Scopus subject areas

  • Cell Biology

Cite this

@article{b8fd7a7035f44404acf407113dd18766,
title = "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",
abstract = "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.",
keywords = "ES cells, Gene targeting, Homologous recombination, HPRT, p53, Rad51",
author = "Juan Dominguez-Bendala and Helen Priddle and Alan Clarke and Jim McWhir",
year = "2003",
month = "6",
day = "10",
doi = "10.1016/S0014-4827(03)00111-3",
language = "English",
volume = "286",
pages = "298--307",
journal = "Experimental Cell Research",
issn = "0014-4827",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - 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

AU - Dominguez-Bendala, Juan

AU - Priddle, Helen

AU - Clarke, Alan

AU - McWhir, Jim

PY - 2003/6/10

Y1 - 2003/6/10

N2 - 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.

AB - 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.

KW - ES cells

KW - Gene targeting

KW - Homologous recombination

KW - HPRT

KW - p53

KW - Rad51

UR - http://www.scopus.com/inward/record.url?scp=18544410292&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=18544410292&partnerID=8YFLogxK

U2 - 10.1016/S0014-4827(03)00111-3

DO - 10.1016/S0014-4827(03)00111-3

M3 - Article

C2 - 12749858

AN - SCOPUS:18544410292

VL - 286

SP - 298

EP - 307

JO - Experimental Cell Research

JF - Experimental Cell Research

SN - 0014-4827

IS - 2

ER -