Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome
Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome
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DOI：
10.1073/pnas.1321889111
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发表时间：
2014-05-27
影响因子：
11.1
通讯作者：
Aplan, Peter D.
中科院分区：
文献类型：
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作者：
Onozawa, Masahiro;Zhang, Zhenhua;Aplan, Peter D.
我们使用I-SceI内切酶产生DNA双链断裂（DSBs），并观察到这些DSBs的一部分通过序列插入修复，我们称之为“模板序列插入”（TSIs），来自基因组的远端区域。这些tsi来源于基因、反转录转座子或端粒序列，并且没有从基因组的供体位点删除，这导致了它们来源于逆转录RNA的假设。RNA和I-SceI表达载体共转染后，在DNA-DSB位点插入RNA衍生序列，tsi被逆转录酶抑制剂抑制。这两个观察结果都支持tsi来自RNA模板的假设。此外，在转录激活因子样效应核酸酶蛋白诱导的DNA dsb位点上检测到类似的插入。骨髓瘤细胞系的全基因组测序显示了额外的tsi，表明通过插入修复DNA dsb并不局限于实验产生的DNA dsb。对公开可用数据库的分析显示，许多这些tsi在人类基因组中是多态的。综上所述，这些结果表明，在全基因组序列数据的解释中，插入事件应被视为总体染色体重排的替代方法，并且这种致突变形式的DNA修复可能在遗传疾病、外显子洗选和哺乳动物进化中发挥作用。
We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.