Altered DNA repair and recombination responses in mouse cells expressing wildtype or mutant forms of RAD51.
Rad51, a homolog of Esherichia coli RecA, is a DNA-dependent ATPase that binds cooperatively to single-stranded DNA forming a nucleoprotein filament, which functions in the strand invasion step of homologous recombination. In this study, we examined DNA repair and recombination responses in mouse hybridoma cells stably expressing wildtype Rad51, or Walker box lysine variants, Rad51-K133A or Rad51-K133R, deficient in ATP binding and ATP hydrolysis, respectively. A unique feature is the recovery of stable transformants expressing Rad51-K133A. Augmentation of the endogenous pool of Rad51 by over-expression of transgene-encoded wildtype Rad51 enhances cell growth and gene targeting, but has minimal effects on cell survival to DNA damage induced by ionizing radiation (IR) or mitomycin C (MMC). Whereas expression of Rad51-K133A impedes growth, in general, neither Rad51-K133A nor Rad51-K133R significantly affected survival to IR- or MMC-induced damage, but did significantly reduce gene targeting. Expression of wildtype Rad51, Rad51-K133A or Rad51-K133R did not affect the frequency of intrachromosomal homologous recombination. However, in both gene targeting and intrachromosomal homologous recombination, wildtype and mutant Rad51 transgene expression altered the recombination mechanism: in gene targeting, wildtype Rad51 expression stimulates crossing over, while expression of Rad51-K133A or Rad51-K133R perturbs gene conversion; in intrachromosomal homologous recombination, cell lines expressing wildtype Rad51, Rad51-K133A or Rad51-K133R display increased deletion formation by intrachromosomal homologous recombination. The results suggest that ATP hydrolysis by Rad51 is more important for some homologous recombination functions than it is for other aspects of DNA repair.
Rukść
Ania
A
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Birmingham
Erin C
EC
Baker
Mark D
MD
D000818:Animals; D004260:DNA Repair / Q000502:physiology*; D018390:Gene Targeting; D051379:Mice; D016297:Mutagenesis, Site-Directed; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011995:Recombination, Genetic / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse