The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase
The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase
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DOI：
10.1074/jbc.272.29.18425
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Richardson, CC
中科院分区：
文献类型：
--
作者：
Notarnicola, SM;Mulcahy, HL;Richardson, CC
噬菌体T7的基因4蛋白在复制叉处提供引发酶和解旋酶活性。有效的DNA复制需要基因4蛋白的功能与T7 DNA聚合酶的运动协调。我们表明，基因4蛋白的羧基末端结构域是必需的与T7 DNA聚合酶在前导链DNA合成的相互作用。基因4蛋白的羧基末端是高度酸性的：在17个羧基末端氨基酸中，7个带负电荷。这17个残基的编码区的缺失导致不能支持T7噬菌体生长的基因4蛋白。纯化的突变体基因4蛋白具有野生型水平的解旋酶和引发酶活性;然而，通过T7 DNA聚合酶在双链体DNA底物上催化的DNA合成被该突变体蛋白刺激至仅为用野生型蛋白获得的合成水平的约5%。突变基因4蛋白可以形成六聚体并结合单链DNA，但如通过非变性PAGE分析所确定的，该蛋白不能与DNA聚合酶形成稳定的复合物。突变基因4蛋白可以正常引发DNA合成，这表明对于滞后链合成，涉及不同的解旋酶/引发酶-DNA聚合酶相互作用。这些发现对T7复制叉上的前导链和滞后链DNA合成的耦合机制具有重要意义。
The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wildtype protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.