Thomas D C, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7744-8. doi: 10.1073/pnas.90.16.7744.
We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.
我们检测了人细胞提取物中紫外线照射的双链DNA在猴病毒40原点依赖性复制的效率和保真度。以噬菌体M13mp2 DNA中大肠杆菌lacZ基因的α-互补结构域作为突变靶点，HeLa细胞提取物中未受损DNA的复制高度准确，而紫外线（280 - 320nm）照射的DNA复制效率较低且准确性较差。复制受到照射的剂量依赖性抑制。尽管如此，仍产生了共价闭合的单体长度环状产物，其对Dpn I消化具有抗性，表明它们是由半保留复制产生的。这些产物可被T4内切核酸酶V切割，而未受损的复制产物则不能，这表明嘧啶二聚体在复制过程中被绕过。当紫外线照射的DNA被复制后用于转染大肠杆菌α-互补宿主菌株以对突变的M13mp2噬菌斑进行评分时，突变噬菌斑频率显著高于未照射、复制的DNA或未复制、紫外线照射的DNA所获得的频率。与紫外线照射相关的诱变率增加和复制抑制在复制前用光解酶处理照射的DNA后均被逆转。对紫外线照射的DNA复制产生的突变体进行序列分析表明，大多数突变体在二嘧啶位点含有C→T转换错误。少数突变体含有1个核苷酸的移码错误或串联双CC→TT替换。这些数据与以下解释一致：在人细胞提取物中，多蛋白复制装置在复制过程中绕过了嘧啶二聚体，并且这种绕过主要通过在二聚体中胞嘧啶（或尿嘧啶）对面错误掺入dAMP而具有诱变性。
