Gene conversion tracts from double-strand break repair in mammalian cells
Gene conversion tracts from double-strand break repair in mammalian cells
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DOI：
10.1128/mcb.18.1.93
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发表时间：
1998-01-01
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;Richardson, C;Jasin, M
哺乳动物细胞能够通过同源重组和通过需要很少或不需要同源性的机制来修复染色体双链断裂（DSB）。虽然自发同源重组是罕见的，但是当同源性由基因靶向实验中的外源DNA或由重复的染色体序列提供时，DSB将刺激重组2至3个数量级，使用小鼠胚胎干细胞中的基因靶向测定，我们现在研究异质性对DSB重组修复的影响。细胞与内切核酸酶表达质粒共转染以诱导染色体DSB，并与含有高达1.2%异质性的底物共转染以修复DSB，我们发现异源性降低了重组修复的效率，1.2%的序列差异导致重组减少了大约六倍。在80个重组体中检查了基因转换区的长度，观察到相对较短的基因转换区，80%的重组体具有58 bp或更少的基因转换区，这些结果表明，哺乳动物细胞中的染色体末端在重组之前通常被保护免于广泛降解。长的基因转换片段（高达511 bp）是连续的，即，它们含有不间断地掺入的沉默突变。这种连续性表明，这些长片段是由末端的广泛降解或异源双链DNA的形成引起的，该异源双链DNA在未断裂链的方向上具有强烈的偏向性。
Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology, Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence, Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs, Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs, We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination, Gene conversion tract lengths were examined in 80 recombinants, Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less, These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.