大肠杆菌uvrD基因产物的体外互补分析。
An in vitro complementation assay for the Escherichia coli uvrD gene product.
作者信息
Kuemmerle N B, Masker W E
出版信息
Nucleic Acids Res. 1983 Apr 11;11(7):2193-204. doi: 10.1093/nar/11.7.2193.
An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.
已开发出一种针对大肠杆菌uvrD基因产物的体外检测方法。该检测方法源自体内实验所揭示的uvrD突变体的特性，其基于在紫外线诱导损伤的切除修复过程中，功能性UvrD蛋白对于共价闭合环状DNA完全重新连接的必要性。当从其他修复缺陷细胞提取物或部分纯化的蛋白质组分中提供功能性UvrD蛋白时，从轻度裂解的uvrD101突变体细胞制备的提取物能够将紫外线损伤的DNA恢复为其共价闭合环状形式。该检测方法用于监测分级分离几个步骤后UvrD蛋白的活性。部分纯化的UvrD蛋白不能补充缺乏DNA聚合酶I或DNA连接酶温度敏感的提取物；然而，它确实能补充来自uvrE和recL位点突变菌株的提取物，这些位点被认为与uvrD位点等位。
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