Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.
Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.
复制标题
小鼠和人类培养细胞中 R 带、G 带和 C 带 DNA 的修复率。
DOI：
10.1159/000077464
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Holmquist,GP
中科院分区：
文献类型：
--
作者：
Sanders,MH;Bates,SE;Wilbur,BS;Holmquist,GP
培养的新生人成纤维细胞和Mus spretus × M.通过用T4核酸内切酶V环嘧啶二聚体糖基化酶切割、碱性琼脂糖凝胶电泳和Southern印迹法分析了经UV照射后的Castaneus F1新生皮肤成纤维细胞。依次用32 P标记的Alu或B2探测印迹，以优先照射R带DNA，用L1探测印迹以优先照射G带DNA，用卫星DNA探测印迹以照射C带DNA。这三个不同的DNA群体表现出略有不同的整体核苷酸切除修复速率的顺序是R带DNA> G带DNA> C带DNA。来自远交新生小鼠和人类的成纤维细胞显示出相似的条带特异性修复率比率，并且鼠成纤维细胞的整体修复率几乎与人成纤维细胞的修复率一样快。进一步分析了人体探针探测信号的质量分布。将凝胶迁移率数据拟合至逻辑方程以包括所有Mr值。随机切割成特定数均分子量的DNA的假设分布与逻辑斯谛凝胶迁移率函数拟合，以确定特定切割频率的这种随机切割分布将如何沿实验凝胶沿着显示。这揭示了一个快速修复的动力学部分，代表17%的L1探测信号（R带DNA），几乎没有L1探测信号（G带DNA），并反映了转录偶联修复的活性基因。其余的紫外线探测的DNA显示了一个随机分布的紫外线诱导的CPD在整个全球核苷酸切除修复的所有阶段。探测的CPD消失，与一级动力学非常吻合，半衰期为7小时。
Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus× M. castaneus F 1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32 P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA. These three different DNA populations showed slightly different global nucleotide excision repair rates that are in the order of speed, R-band DNA> G-band DNA> C-band DNA. Fibroblasts from out-bred neonatal mice and humans showed similar band-specific repair rate ratios and the global repair rate of murine fibroblasts was almost as rapid as that of the human fibroblasts. The mass distribution of the human Alu-probed signal was further analyzed. Gel mobility data was fitted to a logistic equation to include all M r values. Hypothetical distributions of DNA randomly cleaved to a particular number-average molecular weight were fit to the logistic gel mobility function to determine how such a randomly cleaved distribution of a particular cleavage frequency would be displayed along the experimental gel. This revealed a rapidly repaired kinetic fraction that represented 17% of the Alu-probed signal (R-band DNA), almost none of the L1 probed signal (G-band DNA), and reflects transcription coupled repair of active genes. The remaining Alu-probed DNA showed a random distribution of UVC-induced CPDs throughout all stages of global nucleotide excision repair. The Alu-probed CPDs disappeared with an excellent fit to first order kinetics and with a half-life of seven hours.