Ensch-Simon I, Burgers P M, Taylor J S
Department of Chemistry, Washington University, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Jun 2;37(22):8218-26. doi: 10.1021/bi972460j.
The key step in skin cancer induction by UV light is thought to be the mutagenic DNA synthesis past a DNA photoproduct in a proto-oncogene or tumor suppressor gene. To investigate this critical step, we have constructed an SV40 vector containing a cis-syn thymine dimer, the major DNA photoproduct induced by UVB light, within an AseI site at a location that would initially be replicated by leading strand synthesis. When the dimer-containing SV40 vector was incubated with cell-free HeLa extracts in the presence of TAg, and then digested with AseI, a 2325 bp fragment corresponding to inhibition of cleavage at the dimer site was observed, suggesting that the dimer had terminated synthesis and/or had been bypassed. When the reaction was limited to one round of replication and the products of restriction enzyme digestion were examined by denaturing gel electrophoresis, bands corresponding to both termination and bypass were observed in roughly a one-to-one ratio. Whereas increasing the dNTP concentration from 10 microM to 1 mM increased the ratio of bypass to termination from 0.6 to 2.6, it had no effect on the site of termination, which occurred exclusively one nucleotide before the dimer. Experiments in which dGTP was held constant at 25 microM and various combinations of the remaining nucleotides were raised from 25 microM to 1 mM showed substantial increases in the bypass-to-termination ratio, with the greatest effect seen for raising all three nucleotides to 1 mM. Replication by primary fibroblast XPV extracts was also investigated and found to be greatly stimulated by rhRPA, whereas the stimulatory effect for HeLa cell extracts was variable. In the presence of rhRPA, the XPV extracts were also found to bypass the cis-syn dimer, which contrasts with a recent report that could not detect dimer bypass in SV40 transformed XPV extracts in the absence of added replication factors [Cordeiro-Stone, M., et al. (1997) J. Biol. Chem. 272, 13945-13954].
紫外线诱导皮肤癌的关键步骤被认为是在原癌基因或肿瘤抑制基因中，越过DNA光产物进行的致突变性DNA合成。为了研究这一关键步骤，我们构建了一个SV40载体，该载体在一个AseI位点内含有一个顺式胸腺嘧啶二聚体，这是UVB光诱导产生的主要DNA光产物，其位置最初会通过前导链合成进行复制。当将含有二聚体的SV40载体与无细胞的HeLa提取物在TAg存在下孵育，然后用AseI消化时，观察到一个2325 bp的片段，这对应于二聚体位点处切割的抑制，表明二聚体终止了合成和/或被越过。当反应限于一轮复制，并通过变性凝胶电泳检查限制性内切酶消化产物时，观察到对应于终止和越过的条带，其比例大致为一对一。将dNTP浓度从10μM增加到1 mM，越过与终止的比例从0.6增加到2.6，但对终止位点没有影响，终止位点仅在二聚体前一个核苷酸处出现。将dGTP固定在25μM，其余核苷酸的各种组合从25μM增加到1 mM的实验表明，越过与终止的比例大幅增加，将所有三种核苷酸都增加到1 mM时效果最为显著。还研究了原代成纤维细胞XPV提取物的复制，发现rhRPA对其有很大的刺激作用，而对HeLa细胞提取物的刺激作用则各不相同。在rhRPA存在下，还发现XPV提取物越过了顺式二聚体，这与最近一份报告形成对比，该报告在没有添加复制因子的情况下，在SV40转化的XPV提取物中未检测到二聚体越过现象[科尔德罗 - 斯通，M.等人（1997年）《生物化学杂志》272卷，13945 - 13954页]。
