Reconstitution of human DNA polymerase δ using recombinant baculoviruses -: The p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme
Reconstitution of human DNA polymerase δ using recombinant baculoviruses -: The p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme
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DOI：
10.1074/jbc.m109684200
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发表时间：
2002-02-08
影响因子：
4.8
通讯作者：
Fanning, E
中科院分区：
文献类型：
--
作者：
Podust, VN;Chang, LS;Fanning, E
真核DNA聚合酶5被认为由三个（芽殖酵母）或四个亚基（裂殖酵母，哺乳动物）组成。编码多肽p125、p50、p66和p12的四种人类基因已被指定为DNA聚合酶5的亚基。然而，从天然来源中严格纯化人或牛DNA聚合酶5通常产生仅含有p125和p50多肽的双亚基制剂。为了重建完整的DNA聚合酶8，我们构建了编码p125，p50，p66和p12亚基的重组杆状病毒。从用四种杆状病毒感染的昆虫细胞中，分离出含有预期大小的四种多肽的蛋白质制剂。四亚基DNA聚合酶5显示出与从天然来源分离的人、牛和裂殖酵母蛋白质的比活性相当的比活性。重组DNA聚合酶5在复制蛋白A、增殖细胞核抗原和复制因子C的存在下有效复制单引物M13 DNA，并在SV 40 DNA复制系统中具有活性。还分离出由p125、p50和p66亚基组成的三亚基亚复合物，但缺乏p12亚基。p125，p50和p66多肽形成了一个稳定的复合物，显示DNA聚合活性比四亚基聚合酶低15倍。单独表达和纯化的p12对poly（dA）-oligo（dT）模板-引物的三亚基复合物的活性有4倍的刺激作用，但对四亚基酶的活性没有影响。因此，需要p12亚基来重建完全活性的重组人DNA聚合酶5。
Eukaryotic DNA polymerase 5 is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase 5. However, rigorous purification of human or bovine DNA polymerase 5 from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase 8, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase 5 displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase 5 efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase 5.