Lutze L H, Cleaver J E, Morgan W F, Winegar R A
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143-0750.
Mutat Res. 1993 May;299(3-4):225-32. doi: 10.1016/0165-1218(93)90099-y.
DNA double-strand breaks are considered to be the most deleterious lesion induced by ionizing radiation. However, the mechanism of rejoining of these lesions has not been extensively studied at the molecular level. We have used a shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA double-strand breaks in human cells. The advantage of this vector system is that, unlike many previously described shuttle vectors, it has a large target gene for the detection of deletions and it is maintained as a freely replicating episome with chromatin conformation in the nucleus of human cells. In this study we compare data obtained on the spectrum of mutations induced in pHAZE by ionizing radiation (alpha-particles) and restriction enzymes (PvuII, ClaI, and PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand breaks in DNA with known end structures at defined locations and therefore provide a model system for analyzing cellular responses to DNA double-strand breaks. Exposure of human cells containing the vector to alpha-particle irradiation produced both point mutations and large deletions in pHAZE. When the junction regions of the deletions were sequenced it was found that 65% were rejoined with up to 6 bp of homology at the junction region. Analysis of restriction-enzyme-induced mutations suggests that double-strand break ends are modified to facilitate rejoining and that the type of modification is characteristic for different end structures. Double-strand breaks with cohesive ends appear to have fewer modifications introduced at the break points before rejoining than breaks with blunt ends. When considered in relation to the data obtained with ionizing radiation this suggests that the presence of cohesive sequences either at, or in proximity to, the ends enhances rejoining of DNA double-strand breaks.
DNA双链断裂被认为是电离辐射诱导产生的最具危害性的损伤。然而，这些损伤的重新连接机制在分子水平上尚未得到广泛研究。我们使用了一种穿梭载体pHAZE来分析人类细胞中DNA双链断裂的重新连接机制。该载体系统的优势在于，与许多先前描述的穿梭载体不同，它有一个用于检测缺失的大靶基因，并且作为一种自由复制的附加体在人类细胞核中以染色质构象维持。在本研究中，我们比较了电离辐射（α粒子）和限制酶（PvuII、ClaI和PvuI）在pHAZE中诱导的突变谱所获得的数据。与电离辐射不同，限制酶在DNA中已知末端结构的特定位置诱导双链断裂，因此提供了一个用于分析细胞对DNA双链断裂反应的模型系统。将含有该载体的人类细胞暴露于α粒子辐射下，在pHAZE中产生了点突变和大的缺失。当对缺失的连接区域进行测序时，发现65%的缺失在连接区域以高达6个碱基对的同源性重新连接。对限制酶诱导突变的分析表明，双链断裂末端会被修饰以促进重新连接，并且修饰类型对于不同的末端结构具有特征性。具有黏性末端的双链断裂在重新连接之前似乎比平端断裂在断裂点处引入的修饰更少。结合电离辐射获得的数据来看，这表明末端或其附近存在黏性序列会增强DNA双链断裂的重新连接。
