Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by h omologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-s trand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repa ir of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lin es differing solely in the orientation of the repeats. The combination of t he three recombination-test substrates used discriminated among SSA, intrac hromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. G ene conversion with crossover, indistinguishable from long tract gene conve rsion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockou t cells revealed direct evidence for a role of mRAD54 in DSB repair. The su bstrate measuring SSA showed an increased efficiency of DSB repair in the a bsence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was re duced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes g ene conversion with predominant use of the sister chromatid as the repair t emplate at the expense of error-prone SSA.