Seki S, Arakaki Y, Oda T
Department of Biochemistry, Cancer Institute, Okayama University Medical School, Japan.
Acta Med Okayama. 1989 Apr;43(2):73-80. doi: 10.18926/AMO/30873.
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.
建立了一种用于研究参与博来霉素损伤DNA修复的引发因子的简单无细胞系统。用于修复DNA合成的模板引物是通过用2.2微摩尔的博来霉素和20微摩尔的亚铁离子处理pUC19质粒DNA的闭环超螺旋形式制备的。通过博来霉素处理将单链断裂引入pUC19 DNA中，结果DNA大部分转化为开环形式。用引发因子、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用于修复这种博来霉素损伤DNA的系统。孵育后，通过琼脂糖凝胶电泳和电子显微镜分析DNA的构象。开环DNA大部分转化为闭环DNA，表明DNA的单链断裂得到了修复。当省略引发因子时，DNA修复未发生。本系统似乎适用于研究不仅由博来霉素而且由电离辐射或活性氧引起的单链断裂DNA修复中涉及的引发因子。
