Roberts J D, Izuta S, Thomas D C, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1994 Jan 21;269(3):1711-7.
We have measured the fidelity of leading and lagging strand DNA replication in HeLa cell extracts. Providing an excess of one dNTP in reactions induces replication errors consistent with misincorporation of that dNTP. With excess dTTP, both substitutions and single-nucleotide frameshifts are induced. Error distribution is nonrandom; reproducible hot spots for a substitution and a frameshift error are observed. Measurements with two vectors having the origin of replication on opposite sides of the mutational target demonstrate that error rates for G.dTTP and C.dTTP mispairs depend on whether the strand is replicated as the leading or lagging strand. Also, the two hot spots are only observed in one origin-target orientation. Replication reactions reconstituted from two fractions derived from extracts are 3-fold less accurate, but the error specificity with excess dTTP is similar to that with extracts. This suggests that the processes responsible for the nonrandom error rates are not lost as a result of fractionation. Furthermore, the reconstituted system is devoid of mismatch repair activity. Thus, mismatch repair is not responsible for the mispair-, site-, and strand-specific differences observed.
我们已经测量了HeLa细胞提取物中前导链和后随链DNA复制的保真度。在反应中提供过量的一种脱氧核苷酸三磷酸（dNTP）会诱导与该dNTP错掺入一致的复制错误。当有过量的dTTP时，会诱导替换和单核苷酸移码。错误分布是非随机的；观察到了替换和移码错误的可重复热点。使用两个在突变靶点两侧具有复制起点的载体进行测量表明，G·dTTP和C·dTTP错配的错误率取决于该链是作为前导链还是后随链进行复制。此外，仅在一种起点-靶点方向上观察到这两个热点。从提取物衍生的两个组分重建的复制反应准确性降低了3倍，但过量dTTP时的错误特异性与提取物相似。这表明导致非随机错误率的过程不会因分级分离而丧失。此外，重建系统缺乏错配修复活性。因此，错配修复与所观察到的错配、位点和链特异性差异无关。
