Allen Chris, Miller Cheryl A, Nickoloff Jac A
Department of Molecular Genetics and Microbiology, School of Medicine, University of New Mexico, Albuquerque, NM 87131, USA.
DNA Repair (Amst). 2003 Oct 7;2(10):1147-56. doi: 10.1016/s1568-7864(03)00139-3.
In eukaryotes, DNA double-strand breaks (DSBs) are repaired by competing HR and non-homologous end-joining (NHEJ) pathways. DSB repair by HR is highly accurate, while NHEJ can result in deletions and insertions. Transcription enhances certain DNA repair pathways and spontaneous homologous recombination (HR). As a means to promote accurate repair in active genes, we thought it possible that the balance between HR and NHEJ would be shifted toward HR in highly transcribed regions. We tested this idea by examining products of DSB repair in integrated neo-direct repeats under conditions of low-level constitutive, or high-level induced transcription regulated by the dexamethasone (Dex)-responsive mouse mammary tumor virus (MMTV) promoter. DSBs were introduced into one copy of neo by expressing I-SceI nuclease, and DSB repair products were isolated and characterized with an efficient, non-selective assay. We found that transcription does not significantly change the relative frequencies of HR and NHEJ, the relative frequencies of sequence capture and gross chromosomal rearrangement, nor the average size of deletions. About one-third of DSB repair products showed large-scale rearrangements, indicating that a single DSB in a mammalian chromosome has significant mutagenic potential.
在真核生物中，DNA双链断裂（DSB）通过相互竞争的同源重组（HR）和非同源末端连接（NHEJ）途径进行修复。HR介导的DSB修复高度精确，而NHEJ可能导致缺失和插入。转录可增强某些DNA修复途径以及自发同源重组（HR）。作为在活跃基因中促进精确修复的一种方式，我们认为在高度转录区域，HR和NHEJ之间的平衡可能会向HR倾斜。我们通过检测在地塞米松（Dex）应答性小鼠乳腺肿瘤病毒（MMTV）启动子调控的低水平组成型或高水平诱导转录条件下，整合的新霉素直接重复序列中DSB修复的产物，来验证这一想法。通过表达I-SceI核酸酶将DSB引入新霉素的一个拷贝中，并通过高效、非选择性检测方法分离和鉴定DSB修复产物。我们发现转录不会显著改变HR和NHEJ的相对频率、序列捕获和染色体大片段重排的相对频率，也不会改变缺失的平均大小。大约三分之一的DSB修复产物显示出大规模重排，这表明哺乳动物染色体中的单个DSB具有显著的诱变潜力。
