Berdis A J, Soumillion P, Benkovic S J
Pennsylvania State University, Department of Chemistry, University Park 16802-6300, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12822-7. doi: 10.1073/pnas.93.23.12822.
To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated delta C6 exo-, is identical to wild-type exo- polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo- polymerase, the delta C6 exo- polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo- polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein. On the contrary, the peptide appears to disrupt the interaction of the 44/62 protein with the 45 protein, suggesting that the 44/62 protein and the polymerase use the same site on the 45 protein for functional interactions. Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44/62 protein and polymerase.
为了进一步阐明噬菌体T4全酶形成的机制和动力学，构建了一种缺失最后六个羧基末端氨基酸的突变聚合酶，将其过量表达并纯化至同质。这种被命名为δC6 exo-的突变聚合酶，在催化常数、聚合酶活性常数以及核苷酸和DNA底物的解离常数方面与野生型exo-聚合酶相同。然而，与野生型exo-聚合酶不同的是，δC6 exo-聚合酶无法与45蛋白相互作用形成稳定的全酶。一种与野生型exo-聚合酶羧基末端相对应的合成多肽被作为噬菌体T4 DNA复制的体外抑制剂进行测试。令人惊讶的是，该多肽并非通过破坏聚合酶与45蛋白的相互作用来直接抑制全酶复合物的形成。相反，该多肽似乎破坏了44/62蛋白与45蛋白的相互作用，这表明44/62蛋白和聚合酶在45蛋白上利用相同的位点进行功能相互作用。所呈现的数据将根据一个模型进行讨论，该模型将聚合酶羧基末端的功能与与45蛋白的有效相互作用相关联，同时也涉及45蛋白与44/62蛋白和聚合酶的相互作用。
