Kong D, Nossal N G, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Mar 28;272(13):8380-7. doi: 10.1074/jbc.272.13.8380.
Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.
噬菌体T7基因2.5单链DNA结合蛋白和基因4解旋酶共同促进两个同源DNA分子的配对以及随后的极性分支迁移（Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010 - 2019）。在本报告中，我们表明，一旦两个DNA伙伴之间形成了连接分子，基因2.5蛋白对于链转移的起始或传播并非必需，而连接分子的形成反应仅由基因2.5蛋白介导。一种突变的基因2.5蛋白，即基因2.5 - Δ21C蛋白，在其C末端缺少21个氨基酸残基，无法与基因4蛋白发生物理相互作用。尽管它确实能结合单链DNA并通过同源碱基配对促进连接分子的形成，但基因4解旋酶随后的链转移会受到基因2.5 - Δ21C蛋白的抑制。噬菌体T4基因32蛋白同样会抑制T7基因4蛋白介导的链转移，而大肠杆菌单链DNA结合蛋白则不会。噬菌体T7的63 kDa基因4蛋白也是一种DNA引发酶，因为它在单链DNA上移位时能催化在特定序列处合成寡核苷酸。我们发现，同时进行引物合成对链转移的速率和程度均无显著影响。噬菌体T4基因41解旋酶已被证明在T4基因59解旋酶组装蛋白将解旋酶加载到由T4 UvsX和基因32蛋白形成的连接分子上之后，能催化极性分支迁移（Salinas, F., and Kodadek, T. (1995) Cell 82, 111 - 119）。我们发现，仅基因32蛋白就能在部分单链的同源DNA伙伴之间形成连接分子，并且随后的分支迁移除了需要基因41解旋酶和基因59解旋酶组装蛋白外，还需要这种单链DNA结合蛋白。与链转移反应类似，由T4 DNA聚合酶催化的链置换DNA合成除了需要基因41和59蛋白外，也需要基因32蛋白的存在。
