Reconstitution of complete SV40 DNA replication with purified replication factors.
Reconstitution of complete SV40 DNA replication with purified replication factors.
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发表时间：
1994-04
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通讯作者：
S. Waga;Glenn BauerS;B. Stillman
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作者：
S. Waga;Glenn BauerS;B. Stillman
描述了在从猿猴病毒 40 (SV40) 起源复制 DNA 后，鉴定和纯化 I 型 DNA 所需的人类细胞蛋白。使用这些蛋白质，仅用纯化的 DNA 复制因子重建完整的 SV40 DNA 复制：SV40 大肿瘤抗原 (TAg)、复制蛋白 A (RPA)、DNA 拓扑异构酶 I 和 II、DNA 聚合酶 α-引物酶、复制因子 C (RFC)、增殖细胞核抗原 (PCNA)、DNA 聚合酶 δ、成熟因子 1 (MF1) 和 DNA 连接酶 I。 MF1，5' 到3' 核酸外切酶和 DNA 连接酶 I 均被鉴定为产生共价闭合环状松弛（I 型）DNA 的必需成分。 MF1 可能与其他人先前证明的在人工 DNA 模板上的 DNA 合成过程中发挥作用或与来自 SV40 起源的 DNA 聚合酶 α 结合发挥作用的核酸外切酶相同。结合之前的这些研究，我们的结果表明，MF1 的作用是在滞后链 DNA 合成过程中去除附着在每个冈崎片段上的 RNA 引物。有趣的是，虽然哺乳动物DNA连接酶I在重建的复制系统中发挥作用，但哺乳动物DNA连接酶III没有替代并且噬菌体T4DNA连接酶的功能低效，这表明DNA连接酶I作为真核细胞中的复制DNA连接酶具有特定作用。
The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.