Nikolaishvili-Feinberg N, Cordeiro-Stone M
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, 27599-7525, USA.
Biochemistry. 2001 Dec 18;40(50):15215-23. doi: 10.1021/bi011474t.
In vitro replication assays were used to determine the capacity of HeLa extracts to replicate past one of the two major photoproducts produced by ultraviolet radiation at adjacent thymines in duplex DNA, namely, the cis,syn cyclobutane dimer ([c,s]TT) and the 6-4 pyrimidine-pyrimidone adduct ([6-4]TT). The site-specific photoproduct was placed on the template either to the leading strand or to the lagging strand of nascent DNA with respect to the first fork encountering the lesion during bidirectional replication of closed circular duplex molecules carrying the SV40 origin. Replication products from time-course reactions were fractionated by gel electrophoresis in the presence of ethidium bromide. Recognition and quantification of true translesion synthesis products, i.e., newly synthesized closed circular molecules carrying the photoproduct, were aided by specific substrate modifications (a T:T mismatch in a unique PstI site nearby the photoproduct) and improved assay conditions (internal standard to control for completion of PstI digestion). Extracts from HeLa cells, which express DNA polymerase eta, were competent to replicate past the [c,s]TT on either strand. The efficiency of bypass replication of the [c,s]TT on the template to the leading or the lagging strand was 71% and 67%, respectively. The same extracts demonstrated very low efficiency of translesion synthesis (at most 8-10%) of the [6-4]TT on either template position. Replication-competent cell-free extracts from other human cells were also deficient in the bypass of the [6-4]TT in vitro.
体外复制实验用于确定HeLa细胞提取物越过双链DNA中相邻胸腺嘧啶经紫外线照射产生的两种主要光产物之一的复制能力，即顺式、顺式环丁烷二聚体（[c,s]TT）和6-4嘧啶-嘧啶酮加合物（[6-4]TT）。在携带SV40复制起点的闭环双链分子双向复制过程中，相对于遇到损伤的第一个复制叉，将位点特异性光产物置于新生DNA的模板链上，该模板链可以是前导链，也可以是后随链。在溴化乙锭存在的情况下，通过凝胶电泳对时程反应的复制产物进行分级分离。通过特异性底物修饰（光产物附近独特的PstI位点中的T:T错配）和改进的实验条件（用于控制PstI消化完成的内标），有助于识别和定量真正的跨损伤合成产物，即携带光产物的新合成闭环分子。表达DNA聚合酶η的HeLa细胞提取物能够越过任一条链上的[c,s]TT进行复制。模板链上的[c,s]TT在前导链或后随链上的跨损伤复制效率分别为71%和67%。相同的提取物在任一模板位置上对[6-4]TT的跨损伤合成效率都非常低（最多8-10%）。来自其他人类细胞的具有复制能力的无细胞提取物在体外对[6-4]TT的跨损伤复制也存在缺陷。
