Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells
Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells
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DOI：
10.1128/mcb.21.8.2671-2682.2001
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发表时间：
2001-04-01
影响因子：
5.3
通讯作者：
Jasin, M
中科院分区：
文献类型：
--
作者：
Elliott, B;Jasin, M
染色体双链断裂（DSB）在哺乳动物细胞（包括鼠胚胎干（ES）细胞）中以几个数量级刺激同源重组，但重组效率随着修复底物之间的异质性增加而降低（B，Elliott，C，Richardson，J，Winderbaum，J，A. Nickoloff，and M，Jasin，Mel. Cell，Biol.18：93-101，1998），我们现在检查了错配修复（MMR）缺陷ES细胞中的同源重组，以研究重组频率和事件结果。使用在msh 2基因中具有靶向突变的细胞，我们发现对于基因靶向和染色体内重组，趋异底物之间的重组障碍是放松的。因此，具有1.5%趋异的底物在Msh 2（-/-）细胞中经历DSB促进的重组的可能性比在野生型细胞中高10倍。尽管突变细胞可以有效地修复DSB，但重组体中基因转换束的检查表明，它们不能有效地校正与DSB相邻形成的错配异源双链DNA（hDNA）。因此，与来自野生型细胞的重组体相比，>20倍多的来自突变细胞的重组体具有未校正的束。结果表明，哺乳动物细胞中DSB的基因转换修复通常涉及hDNA的错配校正，而不是双链间隙的形成。因此，在MMR缺陷的细胞中，异常重组修复可能是导致基因组不稳定和可能的肿瘤发生的另一种机制。
Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B, Elliott, C, Richardson, J, Winderbaum, J, A. Nickoloff, and M, Jasin, Mel. Cell, Biol. 18:93-101, 1998), We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB, As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.