A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA.
A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA.
复制标题
一种用于研究参与博来霉素损伤 DNA 修复的启动因子的无细胞系统。
DOI：
10.18926/amo/30873
复制
发表时间：
1989
影响因子：
0.5
通讯作者：
T. Oda
中科院分区：
文献类型：
--
作者：
S. Seki;Y. Arakaki;T. Oda
建立了一种简单的无细胞体系，用于研究参与博莱霉素损伤的DNA修复的启动因子。用2.2微米博莱霉素和20微米亚铁离子处理闭合环状、超螺旋形式的pUC19质粒DNA，制备了用于修复DNA合成的模板-引物。经博莱霉素处理后，pUC19 DNA发生单链断裂，DNA大部分转化为开放的环状结构。用一个启动因子、DNA聚合酶(DNA聚合酶β或DNA聚合酶I的Klenow片段)、ATP、T4 DNA连接酶和四种脱氧核苷三磷酸构建了一个修复该DNA损伤的系统。培养后，用琼脂糖凝胶电泳法和电子显微镜分析DNA的构象。开放的环状DNA大部分转化为闭合的环状DNA，表明DNA的单链断裂得到修复。当忽略启动因子时，DNA修复不发生。本系统可用于研究DNA单链断裂的启动因子，不仅可用于博莱霉素引起的DNA单链断裂修复，也可用于电离辐射或活性氧引起的DNA单链断裂修复的研究。
A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established. The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions. Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form. A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I), ATP, T4 DNA ligase and four deoxynucleoside triphosphates. After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy. The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired. When the priming factor was omitted, DNA repair did not occur. The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen.