ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在继续我们对大肠杆菌噬菌体T4模型系统的研究 用于其中高效的体外DNA复制的双链DNA复制 用T4噬菌体：T4DNA聚合酶编码的纯化蛋白获得 (基因43)、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个基因为解旋酶， 核糖核酸酶H和DNA连接酶。 我们已经使用了带有光活化交联残基的DNA引物来 标记T4DNA复制复合体的5个蛋白质：聚合酶，44/62 和45个聚合酶辅助蛋白，32个单链DNA结合 蛋白。我们已经确定了5个人中每一个人的相对位置 蛋白质和DNA与蛋白质接触的长度。我们发现 该聚合酶结合在引物末端，覆盖5-7个碱基。 双链DNA。聚合酶的有效标记需要所有5种蛋白质 和三磷酸腺苷。当可交联性残基是来自31末端的14-20个碱基时 在引物末端，45个辅助蛋白被标记，但仅在 所有5种蛋白质和三磷酸腺苷的存在。当引物末端为25个碱基时 从可交联性残基来看，45蛋白的标记性很大 减少了。我们的数据表明，44/62复合体位于 聚合酶和45种蛋白质。 编码RNase H的T4基因的鉴定 在落后的斯特兰德。我们已经克隆了编码开放读数的T4DNA 与我们发现的RNaseH的N端序列匹配的帧 先前从T4感染细胞中提纯的。含有该基因的质粒组， 位于基因33的上游，表达一种蛋白质，其大小和RNaseH和 5‘至3’DNA核酸外切酶活性与 噬菌体编码的酶。这个35kd的蛋白质有三个区域，具有显著的 氨基酸序列与5‘至3’核酸外切酶结构域的相似性 E.coliPolI和T7基因6外切酶。 T4DNA聚合酶的基本区域。T4DNA聚合酶有六个区域 它们与真核DNA聚合酶具有序列相似性。至 确定共享和唯一区域的功能，我们正在表征 点突变产生的T4 DNA聚合酶的改变 克隆的基因。

We are continuing our study of the E. coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41 and 61 primase-helicase, RNase H, and DNA ligase. We have used DNA primers bearing photoactivated crosslinking residues to label 5 proteins of the T4 DNA replication complex: polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA binding protein. We have determined the relative positions of each of the 5 proteins and the length of DNA in contact with the proteins. We find that the polymerase is bound at the primer terminus and covers 5-7 bases of duplex DNA. Efficient labeling of the polymerase requires all 5 proteins and ATP. When the crosslinkable residue is 14-20 bases from the 31 end of the primer-terminus, the 45 accessory protein is labeled, but only,in the presence of all 5 proteins and ATP. When the primer-terminus is 25 bases from the crosslinkable residue, labeling of the 45 protein is greatly decreased. Our data suggest that the 44/62 complex resides between the polymerase and the 45 proteins. Identification of a T4 Gene Encoding the RNase H Which Removes RNA Primers on the Lagging Strand. We have cloned the T4 DNA encoding an open reading frame which matches the N-terminal sequence of an RNase H which we previously purified from T4 infected cells. Plasmids containing this gene, located upstream of gene 33, express a protein whose size and RNase H and 5' to 3' DNA exonuclease activities are identical to those of the phage-encoded enzyme. This 35 kd protein has three regions with significant amino acid sequence similarity to those of the 5' to 3' exonuclease domain of E. coli pol I and the T7 gene 6 exonuclease. Essential Regions of T4 DNA Polymerase. T4 DNA polymerase has six regions which share sequence similarity with eukaryotic DNA polymerases. To determine the function of shared and unique regions, we are characterizing altered T4 DNA polymerases produced by site-specific mutagenesis of the cloned gene.