Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line.
Overexpression of the RAD52 epistasis group of gene products is a convenient way to investigate their in vivo roles in homologous recombination (HR) and DNA repair. Overexpression has the further attraction that any associated stimulation of HR may facilitate gene-targeting applications. Rad51p or Rad52p overexpression in mammalian cells have previously been shown to enhance some forms of HR and resistance to ionising radiation, but the effects of Rad52p overexpression on gene targeting have not been tested. Here we show that Rad52p overexpression inhibits gene targeting while stimulating extrachromosomal HR. We also find that Rad52p overexpression affects cell-cycle distribution, impairs cell survival and is lost during extensive passaging. Therefore, we suggest that excess Rad52p can inhibit the essential RAD51-dependent pathways of HR most likely to be responsible for gene targeting, while at the same time stimulating the RAD51-independent pathway thought to be responsible for extrachromosomal HR. The data also argue against Rad52p overexpression as a means of promoting gene targeting, and highlight the limitations of using a single HR assay to assess the overall status of HR.
Yáñez
Rafael J
RJ
Gene Targeting Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
Porter
Andrew C G
AC
D015153:Blotting, Western; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002877:Chromosomes, Human / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004843:Epistasis, Genetic; D005455:Fluorescent Antibody Technique; D018390:Gene Targeting; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D007041:Hypoxanthine Phosphoribosyltransferase; C495153:RAD51 protein, human; D051135:Rad51 Recombinase