Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms.
In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.
Lambert
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S
UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60-68 avenue du Général Leclerc, 92265 Fontenay aux Roses cédex, France.
Lopez
B S
BS
D000818:Animals; D006224:Cricetinae; D003434:Crossing Over, Genetic; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics*; D005720:Gamma Rays; D005785:Gene Conversion; D020043:Genes, Essential; D051379:Mice; D051135:Rad51 Recombinase; D011836:Radiation Tolerance / Q000235:genetics; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse