Collins K L, Russo A A, Tseng B Y, Kelly T J
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
EMBO J. 1993 Dec;12(12):4555-66. doi: 10.1002/j.1460-2075.1993.tb06144.x.
DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.
DNA聚合酶α是真核细胞中唯一能够从头起始DNA链的酶，也是体外启动SV40 DNA复制所必需的。我们克隆了人DNA聚合酶α的70 kDa亚基（以下简称B亚基），并在细菌中作为融合蛋白进行表达。纯化的融合蛋白在溶液中以及T抗原与SV40 DNA复制起点结合时，均能与SV40 T抗原形成稳定的复合物。对B亚基突变形式的分析表明，其N端的240个氨基酸足以介导复合物的形成。B亚基融合蛋白促进了包含T抗原和DNA聚合酶α催化亚基（A亚基）的复合物的形成，这表明它起到连接这两种蛋白质的作用。这些物理相互作用在功能上具有重要意义，因为T抗原刺激DNA聚合酶α催化亚基活性的能力高度依赖于B亚基。我们认为，由B亚基介导的相互作用通过促进起点处的DNA链起始和/或促进随后在滞后链模板上的引物合成及DNA链合成，在SV40 DNA复制中发挥重要作用。该蛋白可能在细胞DNA复制中发挥类似作用。
