Bryant P E, Johnston P J
School of Biological and Medical sciences, University of St. Andrews, Fife, Scotland, UK.
Mutat Res. 1993 May;299(3-4):289-96. doi: 10.1016/0165-1218(93)90105-m.
Restriction endonucleases (RE) can be used to mimic and model the clastogenic effects of ionising radiation. With the development of improved techniques for cell poration: electroporation and recently streptolysin O (SLO), it has become possible more confidently to study the relationships between DNA double-strand breaks (dsb) of various types (e.g. blunt or cohesive-ended) and the frequencies of induced metaphase chromosomal aberrations or micronuclei in cytokinesis-blocked cells. Although RE-induced dsb do not mimic the chemical end-structure of radiation-induced dsb (i.e. the 'dirty' ends of radiation-induced dsb), it has become clear that cohesive-ended dsb, which are thought to be the major type of dsb induced by radiation, are much less clastogenic than blunt-ended dsb. It has also been possible, with the aid of electroporation or SLO to measure the kinetics of dsb in cells as a function of time after treatment. These experiments have shown that some RE (e.g. Pvu II) are extremely stable inside CHO cells and at high concentrations persist and induce dsb over a period of many hours following treatment. Cutting of DNA by RE is thought to be at specific recognition sequences (as in free DNA) although the frequencies of sites in native chromatin available to RE is not yet known. DNA condensation and methylation are both factors limiting the numbers of available cutting sites. Relatively little is known about the kinetics of incision or repair of RE-induced dsb in cells. Direct ligation may be a method used by cells to rejoin the bulk of RE-induced dsb, since inhibitors such as araA, araC and aphidicolin appear not prevent rejoining, although these inhibitors have been found to lead to enhanced frequencies of chromosomal aberrations. 3-Aminobenzimide, the poly-ADP ribose polymerase inhibitor is the only agent that has so far been shown to inhibit rejoining of RE-induced dsb. Data from the radiosensitive xrs5 cell line, where chromosomal aberration frequencies are higher after RE treatments than in their normal parental CHO line, indicates that the xrs dsb repair pathway is involved in the repair of these dsb. We found that cells treated simultaneous with Pvu II and T4 ligase yielded lower levels of chromosomal damage than in the WT parental line indicating that Pvu II induced dsb retain their ability to be blunt-end ligated inside the cell.
限制性内切酶（RE）可用于模拟和构建电离辐射的致断裂效应。随着细胞穿孔技术的改进：电穿孔以及最近的链球菌溶血素O（SLO）的发展，现在更有信心研究各种类型（如平端或粘性末端）的DNA双链断裂（dsb）与胞质分裂阻滞细胞中诱导的中期染色体畸变或微核频率之间的关系。尽管RE诱导的dsb并不模拟辐射诱导的dsb的化学末端结构（即辐射诱导的dsb的“脏”末端），但已经清楚的是，粘性末端dsb被认为是辐射诱导的主要dsb类型，其致断裂性比平端dsb小得多。借助电穿孔或SLO，也有可能测量处理后细胞中dsb随时间变化的动力学。这些实验表明，一些RE（如Pvu II）在CHO细胞内极其稳定，在高浓度下会持续存在，并在处理后的数小时内诱导dsb。RE切割DNA被认为是在特定的识别序列处（如在游离DNA中），尽管天然染色质中可供RE作用的位点频率尚不清楚。DNA浓缩和甲基化都是限制可用切割位点数量的因素。关于细胞中RE诱导的dsb的切割或修复动力学，人们了解得相对较少。直接连接可能是细胞用于重新连接大部分RE诱导的dsb的一种方法，因为诸如阿糖腺苷、阿糖胞苷和阿非科林等抑制剂似乎并不能阻止重新连接，尽管已发现这些抑制剂会导致染色体畸变频率增加。3 - 氨基苯甲酰胺，一种聚ADP核糖聚合酶抑制剂，是迄今为止唯一被证明能抑制RE诱导的dsb重新连接的试剂。来自辐射敏感的xrs5细胞系的数据表明，xrs dsb修复途径参与了这些dsb的修复，在该细胞系中，RE处理后的染色体畸变频率高于其正常亲代CHO细胞系。我们发现，同时用Pvu II和T4连接酶处理的细胞产生的染色体损伤水平低于野生型亲代细胞系，这表明Pvu II诱导的dsb在细胞内仍保持其被平端连接的能力。
