Cordeiro-Stone M, Makhov A M, Zaritskaya L S, Griffith J D
Department of Pathology, University of North Carolina, Chapel Hill, USA.
J Mol Biol. 1999 Jun 25;289(5):1207-18. doi: 10.1006/jmbi.1999.2847.
Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.
电子显微镜（EM）用于观察闭环DNA质粒体外复制的中间体。从嘧啶二聚体旁路复制能力正常（IDH4、HeLa）或缺陷（CTag）的人细胞中制备无细胞提取物。DNA底物要么未受损，要么含有单个顺式、同步胸腺嘧啶二聚体。该损伤位于SV40复制起点中心下游385 bp处，并特意位于新合成DNA前导链的模板中。30分钟反应的产物用补骨脂素和紫外线交联，用限制酶线性化，然后铺展用于EM观察。在由旁路复制能力正常或缺陷的提取物复制的受损分子中检测到延伸的单链DNA区域。这些区域可以被大肠杆菌单链DNA结合蛋白覆盖。从独特的限制位点到单链DNA区域的双链DNA长度与位点特异性二聚体对前导链合成的阻断所预测的长度一致。通过用单切割限制酶线性化的复制产物进行S1核酸酶处理，然后通过凝胶电泳检测诊断片段，证实了这些结果。在明显超过二聚体的复制叉中没有延伸的单链DNA区域，这被视为旁路复制的证据。在由IDH4提取物复制的17%的分子中满足了这些标准。
