ENZYMATIC MECHANISMS OF DNA REPLICATION--THE BACTERIOPHAGE T4 SYSTEM

我们正在继续研究E.大肠杆菌噬菌体T4模型 用于双链体DNA复制系统，其中有效的DNA复制 用T4噬菌体编码的纯化蛋白质实现体外试验：T4 DNA聚合酶（基因43），基因32 DNA螺旋不稳定蛋白， 基因44/62和基因45聚合酶辅助蛋白，基因41， 61和59引物酶-解旋酶、RNA酶H和DNA连接酶。 我们 与NIAMS的Tim Meuser和克雷格海德合作， T4 DNA复制蛋白的结构通过X射线衍射。 T4 RNA酶是一种5'至3'核酸外切酶，其从DNA中去除RNA引物。 DNA复制叉的滞后链，并且是 真核和原核细胞复制和修复的RAD 2家族 核酸酶 T4全长天然形式的晶体结构 RNase H已在2.06埃的最大分辨率下被解析。 Mg 2+存在下，但不存在核酸。 最 保守残基聚集在一起，形成一个大裂缝， Mg 2+在酶的活性位点。 T4 RNase H 结构表明，如何在广泛分离的保守区， 更大的核苷酸切除修复蛋白如人XPG可以 组装成类似于较小复制品的结构 核酸酶 推定活性位点的定点突变 T4 RNase H的残基显示，D19 N突变蛋白几乎 没有核酸酶活性，但继续结合双链DNA。 这 突变蛋白用于T4 RNase H与 它是衬底。 D200 N突变体保留了核酸酶，而D157 N 大大减少了活动。 T4 RNase H受到强烈刺激， 在T4基因32 DNA结合的存在下变得进行性 蛋白 缺失C-末端27的截短的RNase H 形成两个“螺旋”的氨基酸，从5'端进行第一次切割， 结束，但降低了继续降解的能力。 这种截短 酶不受32蛋白的刺激，这表明酶的可能作用。 这个C末端区域 我们正在研究基因59蛋白刺激 通过41解旋酶的DNA解旋，以及依赖于 41和61蛋白质，使用亲和层析，化学 交联和诱变。 晶体结构的解 基因59解旋酶组装蛋白的研究正在进行中。

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, 61, and 59 primase-helicase, RNase H, and DNA ligase. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by x-ray diffraction. T4 RNase is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork, and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms maximal resolution in the presence of Mg2+, but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site of the enzyme. The T4 RNase H structure suggests how the widely separated conserved regions in the larger nucleotide excision repair proteins such as human XPG could assemble into a structure like that of the smaller replication nucleases. Site-directed mutagenesis of the presumptive active site residues of T4 RNase H shows that the D19N mutant protein has almost no nuclease activity, but continues to bind double-stranded DNA. This mutant protein is being used for cocrystallization of T4 RNase H with it substrate. The D200N mutant retains the nuclease, whereas D157N has greatly reduced activity. T4 RNase H is strongly stimulated and becomes processive in the presence of the T4 gene 32 DNA binding protein. A truncated RNase H, which is missing the C-terminal 27 amino acids that form two ` helices, makes the first cut from the 5' end, but has reduced ability to continue degradation. This truncated enzyme is not stimulated by 32 protein, suggesting a possible role for this C-terminal region. We are studying the mechanism by which the gene 59 protein stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins, using affinity chromatography, chemical cross-linking, and mutagenesis. The solution of the crystal structure of the gene 59 helicase assembly protein is in progress.