CRYSTALLOGRAPHIC STUDIES OF THE T7 REPLICATION FORK

DNA复制是由一系列酶催化的，这些酶一起解开DNA，并精确地复制每条DNA链。DNA合成中的错误可能导致基因组DNA的突变和畸形蛋白质的产生，这可能破坏细胞功能并导致癌症等疾病。噬菌体T7是了解DNA复制保真度、速度和机制的结构基础的一个很好的模型系统。整个复制过程由5种蛋白编码：基因5编码的DNA聚合酶，其加工因子大肠杆菌硫氧还蛋白，基因4编码的解旋酶-引物酶的两个亚基，基因2.5单链DNA结合蛋白（gp2.5）。缺少gp2.5后，体外DNA合成速率急剧降低，而gp2.5是DNA在体内复制所必需的，大大提高了前导链和滞后链的合成速度。T7复制系统的简单性为有效研究gp2.5在复制中的个体作用以及它与复制叉的其他蛋白质的相互作用提供了机会。gp2.5的结构将通过单独gp2.5和结合DNA的x射线晶体学研究来研究。复合体的结构将直接揭示与DNA相互作用的模式。Gp2.5与基因5聚合酶-硫氧还蛋白复合物（T7 DNA聚合酶全酶）和基因4解旋酶-引物酶物理相互作用。这些相互作用对于前导链和滞后链合成的耦合至关重要。gp2.5和T7 DNA聚合酶的相互作用将通过超离心分析更精确地表征。沉降平衡实验将直接测量这种蛋白质-蛋白质结合的稳定性和亚基化学计量学。该信息将用于制备该配合物的结晶。gp2.5与基因4解旋酶引物酶的相互作用将通过x射线晶体学研究进行研究。gp2.5-T7 DNA聚合酶全酶复合物或gp2.5-基因4复合物的结构测定将提供关于复制叉上蛋白质之间相互作用的直接信息。

DNA replication is catalyzed by a host of enzymes that work together to unwind the DNA and make an accurate copy of each strand. Errors in DNA synthesis can lead to mutations in genomic DNA and the production of malformed proteins, which can disrupt cellular functions and lead to diseases such as cancer. Bacteriophage T7 is an excellent model system for understanding the structural basis for the fidelity, processivity, and mechanism of DNA replication. The entire replication process is encoded by five proteins: the DNA polymerase encoded by gene 5, its processivity factor E. Coli thioredoxin, two subunits of the helicase- primase encoded by gene 4, and the gene 2.5 single-stranded DNA binding protein (gp2.5). The rate of DNA synthesis in vitro is drastically reduced in the absence of gp2.5, and it is essential for DNA replication in vivo, greatly enhancing the processivity of both leading and lagging strand synthesis. The simplicity of the T7 replication system affords an opportunity to effectively study gp2.5's individual role in replication as well as its interactions with the other proteins of the replication fork. The structure of gp2.5 will be investigated through x-ray crystallographic studies of gp2.5 alone and bound to DNA. The structure of the complex will directly reveal the mode of interaction with DNA. Gp2.5 physically interacts with the gene 5 polymerase-thioredoxin complex (the T7 DNA polymerase holoenzyme) and the gene 4 helicase- primase. These interactions are vital for the coupling of leading and lagging strand synthesis. The interaction of gp2.5 and T7 DNA polymerase will be more precisely characterized by analytical ultracentrifugation. Sedimentation equilibrium experiments will directly measure the stability and subunit stoichiometry of this protein-protein association. This information will be used in preparation for the crystallization of the complex. The interactions of gp2.5 and the gene 4 helicase-primase will be investigated through x-ray crystallographic studies. The determination of the structure of the gp2.5-T7 DNA polymerase holoenzyme complex or the gp2.5-gene 4 complex will provide direct information on the interactions between proteins at the replication fork.