ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

DNA复制的酶促机制--噬菌体T4系统

• 批准号: 3964297
• 负责人: N G NOSSAL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3964297
• 关键词: DNA directed DNA polymerase; Escherichia coli; bacterial genetics; bacteriophage T4; chemical chain length; circular DNA; electron microscopy; endonuclease; enzyme induction /repression; gene expression; microorganism genetics; molecular cloning; nuclease; nucleic acid metabolism; nucleic acid sequence; temperature sensitive mutant; virus genetics

We are studying the E. coli bacteriophage T4 as a model system for duplex DNA replication. Efficient DNA replication in vitro is achieved with seven purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43 product), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, and the genes 41 and 61 priming proteins. 61 protein alone has a limited primase activity and 41 protein alone has a DNA unwinding (helicase) activity. The two proteins together form a functional complex that catalyzes both activities much more efficiently than the individual proteins. Although 61 protein alone synthesizes mainly the dimers pppAC and pppGC, only the rare oligonucleotides which begin with G and are long enough to hybridize stably to the DNA template are able to initiate new DNA chains in the absence of 41 protein, in contrast to the pentanucleotide primers found at the ends of DNA with the 41 and 61 proteins together. On T4 DNA, which has hydroxymethyl cytosine in place of cytosine, DNA chain initiation requires both the 41 and 61 proteins. We have proposed and are currently testing a model in which the interaction of the polymerase-accessory protein complex with the primase-helicase controls the selection of priming sites for the initiation of new fragments on the lagging strand. We have constructed plasmids expressing 41 protein with N-terminal and C-terminal deletions and have purified these proteins to identify 41 protein domains required for its primase, helicase, and DNA-dependent nucleotidase activities. We are studying the factors and sequences regulating the expression of the T4 DNA replication proteins, including genes uvsX (recombination protein) through genes 41, 61, and dam (DNA adenine methylase). We have located two strong transcription promoters 700-800 bp upstream of uvsX which are active in vivo and in vitro. A transcription terminator between genes uvsX and 41 contributes to the differential levels of these two proteins. The T4 dam protein methylates the A in the sequence GATC. Expression of the T4 dam gene in pBR322-based plasmids causes E. coli cells to grow poorly, particularly if they lack RNase H.

我们正在研究大肠杆菌噬菌体T4作为双链的模型系统 DNA复制。在体外实现了高效的DNA复制 T4噬菌体编码的纯化蛋白：T4DNA聚合酶(基因43 产品)、基因32 DNA螺旋不稳定蛋白、基因44/62和基因 45个聚合酶辅助蛋白，41和61个基因启动蛋白。 61蛋白单独具有有限的启动酶活性，而41蛋白单独具有 DNA解旋(解旋酶)活性。这两种蛋白质一起形成一种 更有效地催化这两种活性的功能复合体 而不是单独的蛋白质。虽然61蛋白单独合成主要 二聚体pppAC和pppGC，只有稀有的以 G和足够长的能够与DNA模板稳定杂交的能够 在没有41种蛋白质的情况下启动新的DNA链，而不是 在DNA末端发现的41和61的五核苷酸引物 蛋白质结合在一起。在T4 DNA上，它用羟甲基胞嘧啶代替 胞嘧啶，DNA链的起始需要41和61两种蛋白质。我们 已经提出并正在测试一种模型，在该模型中， 与底物酶-解旋酶对照的聚合酶-辅助蛋白复合体 新片段的起始点的选择 落后的一股。我们已经构建了表达41蛋白的质粒 N-端和C-端缺失，并纯化了这些蛋白以 鉴定其底物酶、解旋酶和蛋白质的41个结构域 依赖DNA的核苷酸酶活性。 我们正在研究调节该基因表达的因素和序列 T4DNA复制蛋白，包括基因uvsX(重组蛋白) 通过基因41、61和DAMA(DNA腺嘌呤甲基酶)。我们已经找到了两个 UvsX上游具有活性的700-800个核苷酸的强转录启动子 在体内和体外。UvsX和41基因之间的转录终止子 导致这两种蛋白质水平的差异。T4大坝 蛋白质使GATC序列中的A甲基化。T4大坝的表现形式 PBR322基质粒中的基因会导致大肠杆菌细胞生长不良， 特别是如果他们缺乏核糖核酸酶H。