Clark J M, Hanawalt P C
Mutat Res. 1984 Jul-Aug;132(1-2):1-14. doi: 10.1016/0167-8817(84)90061-0.
We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.
我们使用猿猴病毒40（SV40）作为探针来研究哺乳动物细胞中紫外线损伤DNA的复制。通过在限制温度下培养对DNA合成起始温度敏感的SV40突变体（tsA58），使感染的猴肾细胞中的病毒DNA复制同步，然后添加阿非迪霉素以在允许温度下暂时抑制DNA合成，同时允许复制前事件发生。去除药物后，将感染的细胞以100 J/m2（254 nm）的剂量进行照射，使每个SV40基因组产生6 - 7个嘧啶二聚体，然后回到限制温度以防止从SV40起始点重新启动复制。复制中间体（RI）用[3H]胸苷标记，并通过在CsCl/溴化乙锭梯度中离心，随后进行BND - 纤维素层析进行分离。照射后不久分离的RI中，子代DNA链的大小分布偏向于长度小于亲代DNA中二聚体间距的方向；这种偏差在照射后至少持续1小时，但在3小时内消失，此时新合成的DNA大小超过了二聚体间距。在复制中间体或I型（闭环）DNA分子中，均未检测到亲代链中二聚体的显著切除。这些数据与以下假设一致：复制叉被在叉的前导链一侧遇到的二聚体暂时阻断，但最终在二聚体相对处建立了子代链的连续性。获得的证据表明，在照射后的后期产生了I型分子，其中子代DNA链含有二聚体。因此，DNA链交换以及跨二聚体合成可能参与了从紫外线损伤的SV40复制中间体产生超螺旋I型DNA的过程。
