Roberts J D, 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. 1991 Apr 15;88(8):3465-9. doi: 10.1073/pnas.88.8.3465.
We have asked whether exonucleolytic proofreading occurs during simian virus 40 origin-dependent, bidirectional DNA replication in extracts of human HeLa cells. In addition, we have compared the fidelity of leading and lagging strand DNA synthesis. In a fidelity assay that scores single-base substitution errors that revert a TGA codon in the lacZ alpha gene in an M13mp vector, providing an excess of a single dNTP substrate over the other three dNTP substrates in a replication reaction generates defined, strand-specific errors. Fidelity measurements with two vectors having the origin of replication on opposite sides of the opal codon demonstrate that error rates for two different A.dCTP and T.dGTP mispairs increase when deoxyguanosine monophosphate is added to replication reaction mixtures or when the concentration of deoxynucleoside triphosphates is increased. The data suggest that exonucleolytic proofreading occurs on both strands during bidirectional replication. Measurements using the two simian virus 40 origin-containing vectors suggest that base substitution error rates are similar for replication of the leading and lagging strands.
我们探讨了在人HeLa细胞提取物中，猴病毒40来源依赖性双向DNA复制过程中是否发生核酸外切校对。此外，我们还比较了前导链和后随链DNA合成的保真度。在一项保真度检测中，通过对M13mp载体中lacZα基因里一个TGA密码子回复的单碱基替换错误进行评分，在复制反应中提供过量的单一dNTP底物而非其他三种dNTP底物，会产生特定的、链特异性的错误。使用两个在乳白密码子两侧具有复制起点的载体进行保真度测量表明，当向复制反应混合物中添加脱氧鸟苷单磷酸或增加脱氧核苷三磷酸的浓度时，两种不同的A·dCTP和T·dGTP错配的错误率会增加。数据表明在双向复制过程中，两条链上均发生核酸外切校对。使用两个含有猴病毒40复制起点的载体进行测量表明，前导链和后随链复制的碱基替换错误率相似。
