ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在研究E。大肠杆菌噬菌体T4作为双链体的模型系统 DNA复制。 在体外实现了有效的DNA复制， 由T4噬菌体编码的纯化蛋白质：T4 DNA聚合酶（基因43 产物）、基因32 DNA螺旋不稳定蛋白、基因44/62和基因44/62。 45聚合酶辅助蛋白，以及基因41和61引发蛋白。 单独的61蛋白具有有限的引发酶活性，单独的41蛋白具有有限的引发酶活性。 DNA解旋（解旋酶）活性。 这两种蛋白质一起形成一个 更有效地催化这两种活动的功能复合物 而不是单个蛋白质。 虽然61蛋白单独合成主要 二聚体pppAC和pppGC，只有开始为 G和足够长以与DNA模板稳定杂交的DNA能够 在没有41蛋白的情况下启动新的DNA链，与之相反， 在DNA末端发现的五核苷酸引物，具有41和61 蛋白质在一起。 在T4 DNA上，它有羟甲基胞嘧啶代替 胞嘧啶，DNA链起始需要41和61蛋白。 我们 已经提出并正在测试一个模型，在这个模型中， 聚合酶-辅助蛋白复合物与引物酶-解旋酶控制 选择引发位点以在细胞上引发新片段， 落后股 我们构建了表达41蛋白的质粒， N-末端和C-末端缺失，并已纯化这些蛋白质， 鉴定了其引发酶、解旋酶所需的41个蛋白质结构域， DNA依赖性核苷酸酶活性。 我们正在研究调控基因表达的因子和序列， T4 DNA复制蛋白，包括基因uvsX（重组蛋白） 通过基因41、61和dam（DNA腺嘌呤甲基化酶）。 我们找到了两个 在uvsX上游700-800 bp处有活性的强转录启动子 在体内和体外。 基因uvsX和41之间的转录终止子 导致了这两种蛋白质的不同水平 T4大坝 蛋白质甲基化序列GATC中的A。 T4母兽的表达 pBR 322质粒中的基因引起E.大肠杆菌细胞生长不良， 特别是当它们缺乏RNase H时。

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.