ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在研究大肠杆菌噬菌体T4作为模型系统 用于双链DNA复制。高效的体外DNA复制是 用T4噬菌体：T4编码的七种纯化蛋白获得 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43聚合酶 辅助蛋白，以及基因41和61启动蛋白。 起始酶-解旋酶。61和41蛋白以复合体的形式发挥作用 具有启动酶和DNA解旋(解旋酶)活性。这个 蛋白质扮演着解旋酶的角色，在新生细胞之前打开双链 前导链，并周期性地停止以制造五环核苷酸 引物需要在滞后的链上启动新的链。我们 表明41-61解旋酶与一个区域相互作用 滞后链模板上大于50个核苷酸和40个 前导链模板上的核苷酸。平仓的速度 通过41/61解旋酶是最大的，与所有四个rNTP需要 底漆合成。 61和41蛋白共同构成了主要的五聚体。 在体内启动新的T4 DNA链的引物。61蛋白 单独有一种弱的底物酶活性，主要是二聚体 以及较长产品的痕迹。在缺乏41种蛋白质的情况下， 基因32单链DNA结合蛋白强烈刺激合成 超长RNA(n超过300)通过高浓度的61 蛋白质。我们的研究表明，41蛋白是特定的 刺激以A开头的五聚体的合成，防止 来自延长五聚体的61和32蛋白质，但是 通过T4 DNA聚合酶促进它们延伸到DNA中，并 它的辅助蛋白质。使用凝胶-DNA延缓试验，我们 研究表明，41-蛋白只与单链DNA在 存在BITH 61蛋白和NTP。捆绑最好的是 ATP或GTP，它们是41蛋白解旋酶的辅助因子。 核糖核酸酶H我们已经证明了核糖核酸酶H活性增加了10倍 在T4感染后，已经从T4感染的人中纯化了这一活性 细胞。这种核糖核酸酶H有效地去除了五聚体引物 由41和61两种蛋白合成。

We are studying the E. coli bacteriophage T4 as a model system for duplex DNA replicatoin. 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 43 polymerase accessory proteins, and the genes 41 and 61 priming proteins. Primase-Helicase. The 61 and 41 proteins function as a complex with primase and DNA-unwinding (helicase) activities. The proteins act as a helicase to open the duplex ahead of the nascent leading strand, and stop periodically to make the pentanocleotide primers needed to initiate new chains on the lagging strand. We have shown that the 41-61 helicase interacts with a region of greater than 50 nucleotides on the lagging strand template and 40 nucleotides on the leading strand template. The rate of unwinding by the 41/61 helicase is greatest with all four rNTP needed for primer synthesis. The 61 and 41 proteins together make mainly the pentamer primers which initiate new T4 DNA strands in vivo. 61 protein alone has a weak primase activity making predominantly dimers and traces of longer products. In the absence of 41 protein, the gene 32 ssDNA binding prtein strongly stimulates the synthesis of very long RNA (n more than 300) by high concentrations of 61 proteins. Our studies suggest that 41 protein specifically stimulates the synthesis of pentamers beginning with A, prevents the 61 and 32 proteins from elongating the pentamer, but facilitates their elongation into DNA by T4 DNA polymerase and its accessory proteins. Using a gel-DNA retardation assay, we have shown that 41-protein binds tightly to ssDNA only in the presence of bith 61 protein and an NTP. Binding is greatest with ATP or GTP, which serve as cofactors for the 41 protein helicase. RNase H. We have shown that RNase H activity increases 10-fold after T4 infection and have purified this activity from T4 infected cells. This RNase H efficiently removes the pentamer primers synthesized by the 41 and 61 proteins.