Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange
Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange
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DOI：
10.1128/mcb.20.9.3147-3156.2000
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发表时间：
2000-05-01
影响因子：
5.3
通讯作者：
Kanaar, R
中科院分区：
文献类型：
--
作者：
Dronkert, MLG;Beverloo, HB;Kanaar, R
细胞可以通过具有或不具有交换的基因转换通过同源重组实现DNA双链断裂（DSB）的无错误修复。相反，另一种同源依赖性DSB修复途径，单链退火（SSA），导致缺失。在这项研究中，我们分析了mRAD 54，一个基因参与同源重组，对修复的位点特异性I-SceI诱导的DSB位于一个重复的DNA序列中的小鼠胚胎干细胞的基因组。我们使用了六个同基因细胞系不同的唯一方向的重复。所用的三种重组试验底物的组合可区分SSA、染色单体内基因转换和姐妹染色单体基因转换。DSB修复对于允许SSA事件恢复的底物是最有效的。与长束基因转换，难以区分的交叉基因转换，优先涉及的姐妹染色单体，而不是在同一染色单体上的重复。比较mRAD 54野生型和敲除细胞中的DSB修复揭示了mRAD 54在DSB修复中的作用的直接证据。测量SSA的底物显示在不存在mRAD 54的情况下DSB修复的效率增加。测量姐妹染色单体基因转换的底物显示有和没有交叉的基因转换减少。与这一观察结果一致，mRAD 54缺陷细胞中DNA损伤诱导的姐妹染色单体交换减少。我们的研究结果表明，mRAD 54促进基因转换，主要使用姐妹染色单体作为修复模板，以牺牲易错SSA为代价。
Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair 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 lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence 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 reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.