Yamanishi D T, Bowden G T, Cress A E
Arizona Cancer Center, Tucson 85724.
Biochim Biophys Acta. 1987 Oct 9;910(1):34-42. doi: 10.1016/0167-4781(87)90092-3.
Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.
用（±）7β，8α-二羟基-9α，10α-环氧-7,8,9,10-二氢苯并[a]芘（BP二醇环氧化物I）处理同步化的中国仓鼠卵巢（CHO）细胞，以测试哺乳动物细胞中复制旁路修复的“阻断-间隙”模型。该模型的一个特点是致癌或致突变的DNA加合物作为前导链上DNA复制叉的阻断物。使用同步化的CHO细胞，通过对新复制的DNA进行BrdUrd标记来测量S期进展速率。当细胞在G1/S边界分别用0.33和0.66μM的BP二醇环氧化物I处理时，S期进展速率分别降低了22%和42%。使用pH阶跃碱性洗脱试验，发现S期进展速率降低是由于多个复制子大小的新生DNA出现延迟。这一观察结果与前导链中存在的BP-DNA加合物的频率一致。“阻断-间隙”模型的另一个特点是，滞后链上的加合物诱导的阻断将产生间隙。通过使用高分辨率琼脂糖凝胶电泳确定，在BP二醇环氧化物I处理的同步化CHO细胞中，冈崎片段大小的复制中间体的连接以剂量依赖的方式被阻断。这些数据与DNA致癌物加合物对DNA复制前导链的阻断一致。BP二醇环氧化物I对冈崎片段大小的片段连接的抑制意味着对DNA滞后链复制的阻断，导致间隙形成。因此，这里给出的数据支持致癌物损伤的哺乳动物细胞中复制旁路修复的“阻断-间隙”模型。
