Honma Masamitsu, Sakuraba Mayumi, Koizumi Tomoko, Takashima Yoshio, Sakamoto Hiroko, Hayashi Makoto
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
DNA Repair (Amst). 2007 Jun 1;6(6):781-8. doi: 10.1016/j.dnarep.2007.01.004. Epub 2007 Feb 12.
DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.
DNA双链断裂（DSBs）通常通过非同源末端连接（NHEJ）或同源重组（HR）进行修复。虽然HR基本上是无差错修复，但NHEJ是一种诱变途径，会导致缺失。NHEJ必须受到精确调控以维持基因组完整性。为了阐明NHEJ的作用，我们研究了人类细胞中DSBs的NHEJ修复的遗传后果。人类淋巴母细胞系TSCE5和TSCE105在内源性胸苷激酶基因（TK）中分别有单个和两个I-SceI内切酶位点，该基因位于17号染色体长臂上。I-SceI的表达在TK基因处产生DSBs。我们使用新型转染系统（Amaxa Nucleofector）将I-SceI表达载体导入细胞并随机分离克隆。我们在3%的TSCE5细胞和30%的TSCE105细胞克隆中发现了TK基因中与DSBs相关的突变。TSCE5中的大多数突变是小（1 - 30bp）缺失，连接处有0 - 4bp的微同源性。其他的包括大（>60）bp缺失、一个插入和一个重排。等位基因间HR产生的突变体也有发生，但频率较低。另一方面，TSCE105中的大多数突变是缺失，这些缺失包含了由NHEJ在DSBs处产生的两个I-SceI位点。序列连接与TSCE5突变体中的相似。有趣的是，一些突变体通过完美连接两个原始I-SceI位点而不删除断裂末端形成了一个新的I-SceI位点。这些结果支持了这样一种观点，即修复I-SceI诱导的DSBs的NHEJ主要导致小缺失或无缺失。因此，NHEJ必须通过修复DSBs以及防止许多有害改变来帮助维持哺乳动物细胞中的基因组完整性。
