Onozawa Masahiro, Zhang Zhenhua, Kim Yoo Jung, Goldberg Liat, Varga Tamas, Bergsagel P Leif, Kuehl W Michael, Aplan Peter D
Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and.
Comprehensive Cancer Center, Mayo Clinic, Scottsdale, AZ 85259.
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7729-34. doi: 10.1073/pnas.1321889111. Epub 2014 May 12.
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.
我们使用I-SceI核酸内切酶产生DNA双链断裂（DSB），并观察到其中一部分DSB通过插入基因组远处区域的序列得以修复，我们将这些序列称为“模板化序列插入”（TSI）。这些TSI源自基因、逆转座子或端粒序列，且未从基因组中的供体位点删除，这引发了一种假设，即它们源自逆转录的RNA。RNA与I-SceI表达载体共转染证明了RNA衍生序列在DNA-DSB位点的插入，并且TSI被逆转录酶抑制剂所抑制。这两个观察结果都支持TSI源自RNA模板的假设。此外，在转录激活样效应核酸酶蛋白诱导的DNA DSB位点也检测到了类似的插入。骨髓瘤细胞系的全基因组测序揭示了更多的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表明，这些TSI中有许多在人类基因组中是多态性的。综上所述，这些结果表明，在解释全基因组序列数据时，插入事件应被视为染色体大片段重排的替代方式，并且这种诱变形式的DNA修复可能在遗传疾病、外显子改组和哺乳动物进化中发挥作用。
