Carty M P, Ishimi Y, Levine A S, Dixon K
Section on Viruses and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Mutat Res. 1990 Oct;232(2):141-53. doi: 10.1016/0027-5107(90)90119-o.
To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.
为了确定DNA聚合酶α对哺乳动物系统中整体DNA复制保真度的贡献，我们在一个重组的体外DNA复制系统中测量了基于SV40的穿梭载体pZ189的复制保真度，该系统包含纯化的HeLa DNA聚合酶α（此外还有单链DNA结合蛋白、拓扑异构酶II、DNA连接酶、5'→3'核酸外切酶、核糖核酸酶H和SV40 T抗原）。我们发现，在该系统中进行双向复制时，DNA聚合酶α具有高度准确性。在重组复制系统中，DNA聚合酶α的这种高复制保真度与在没有其他复制因子的情况下，纯化的HeLa细胞DNA聚合酶α对同一靶基因进行填补缺口DNA合成时相对较低的保真度形成对比。尽管在重组系统中DNA聚合酶α的DNA复制保真度相对较高，但比含有包括DNA聚合酶δ在内的其他复制因子的HeLa细胞粗提物中的DNA复制保真度低约4倍。这些结果表明，在一个最小的重组复制系统中进行半保留DNA复制时，DNA聚合酶α有能力以高保真度复制DNA，但重组系统中不存在的其他细胞因子可能有助于粗提系统更高的复制保真度。
