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 ca rrying tandem repeat sequences with an I-SceI cleavage site, We characteriz ed conservative recombination after I-SceI cleavage as gene conversion or i ntrachromatid crossing over associated with random reintegration of the exc ised reciprocal product. We identified two dominant-negative RAD51 forms th at specifically inhibit conservative recombination: the yeast ScRAD51 or th e yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates co nservative recombination, None of these RAD51 forms affects non-conservativ e recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD 51 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 drastic ally 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.