Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

AUTOR(ES)

Lambert, Sarah

FONTE

Oxford University Press

RESUMO

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-SceI cleavage site. We characterized conservative recombination after I-SceI 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 γ-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.

Documentos Relacionados

• Aberrant Double-Strand Break Repair in rad51 Mutants of Saccharomyces cerevisiae
• In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair
• Modulation of Saccharomyces Cerevisiae DNA Double-Strand Break Repair by Srs2 and Rad51
• Overexpression of human RAD51 and RAD52 reduces double-strand break-induced homologous recombination in mammalian cells
• Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.