Recombination of parent and daughter strand DNA after UV-irradiation in mammalian cells
Recombination of parent and daughter strand DNA after UV-irradiation in mammalian cells
复制标题
哺乳动物细胞中紫外线照射后母链和子链 DNA 的重组
作者：
A. Fornace
哺乳动物细胞复制含有嘧啶二聚体的DNA的机制尚不清楚。当细菌在紫外线照射后开始DNA合成时，含有嘧啶二聚体的亲本DNA已被证明通过依赖reca的机制“交换”到子链DNA中。在早期的实验中，当生长的哺乳动物细胞被紫外线照射，然后用标记的胸腺嘧啶孵育时，嘧啶二聚体最初被解释为在新合成的DNA中4，但后来发现仅在紫外线处理前的子链中与新合成的DNA相邻5。避免这些解释问题的一种方法是使用在照射时不合成DNA的G0或G1细胞。紫外损伤可以通过一种非常灵敏的定量分析来检测，例如最近描述的使用碱性洗脱和从黄体微球菌中提取的核酸内切酶制剂6。我现在使用这种方法，并报告在紫外线照射后34-45小时，在人外周血淋巴细胞（PBL）、正常人成纤维细胞、A组着色性干皮病（XP）成纤维细胞和小鼠3T3细胞中，每109道吨子链DNA可检测到多达3个二聚体。这代表大约1-3%的二聚体存在于母体链在紫外线照射后的这个时间。
The mechanism by which mammalian cells replicate DNA containing pyrimidine dimers is poorly understood. When DNA synthesis is initiated after UV-irradiation in bacteria, parental DNA containing pyrimidine dimers has been shown to ‘exchange’ into the daughter strand DNA by a recA-dependent mechanism1–3. In earlier experiments, when growing mammalian cells were UV-irradiated and then incubated with labelled thymidine, pyrimidine dimers were initially interpreted to be in the newly synthesized DNA4, but later were found to be only adjacent to newly synthesized DNA in daughter strand present before UV treatment5. A way to avoid these problems of interpretation would be to use cells in G0 or G1 which are not synthesizing DNA at the time of irradiation. UV damage could then be detected in a very sensitive quantitative assay such as that recently described using alkaline elution and an endonuclease preparation from Micrococcus luteus6. I have now used this approach and report that up to 3 dimers per 109 daltons of daughter strand DNA could be detected 34–45 h after UV-irradiation in human peripheral blood lymphocytes (PBL), normal human fibroblasts, group A xeroderma pigmentosum (XP) fibroblasts and mouse 3T3 cells. This represents approximately 1–3% of the dimers present in the parent strand at this time after UV.