Veaute X, Mari-Giglia G, Lawrence C W, Sarasin A
Institut de Recherches sur le Cancer, UPR 42 CNRS, Laboratoire de Génétique Moléculaire, BP 8, 7, rue Guy-Moquet, 94801, Villejuif, France.
Mutat Res. 2000 Feb 16;459(1):19-28. doi: 10.1016/s0921-8777(99)00052-x.
DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis, syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.
真核细胞中的DNA复制涉及多种蛋白质，这些蛋白质以不对称协调的方式合成前导链和后随链。为了分析这种不对称性对紫外线诱导损伤的跨损伤合成的影响，我们用含有SV40复制起点的质粒与独特的位点特异性顺式、syn-环丁烷二聚体或嘧啶-嘧啶酮（6-4）光产物一起孵育，该光产物位于前导链或后随链模板上，同时加入由人HeLa细胞制备的具有DNA复制能力的提取物。二维琼脂糖凝胶电泳分析显示，只有当紫外线损伤位于前导链模板上时，才会强烈阻碍叉状结构的前进。由于DNA解旋酶负责在叉状结构之前解开双链DNA，并且是第一个遇到任何潜在损伤的成分，我们测试了这些单一光产物对SV40 T抗原解旋活性的影响，SV40 T抗原是我们体外复制试验中的主要解旋酶。我们表明，在双链DNA底物中，紫外线诱导的DNA损伤不会抑制SV40 T抗原解旋酶的活性。
