CRYSTALLOGRAPHIC STUDIES OF THE T7 REPLICATION FORK

DNA复制是由一系列酶催化的，这些酶共同作用以解开DNA并精确复制每条链。DNA合成中的错误会导致基因组DNA的突变和畸形蛋白质的产生，这会破坏细胞功能并导致癌症等疾病。 噬菌体T7是一个很好的模型系统，用于理解DNA复制的保真度，持续合成能力和机制的结构基础。整个复制过程由五种蛋白质编码：基因5编码的DNA聚合酶，它的持续合成因子E。大肠杆菌硫氧还蛋白，由基因4编码的解旋酶-引发酶的两个亚基，和基因2.5单链DNA结合蛋白（gp2.5）。在没有gp2.5的情况下，体外DNA合成的速率急剧降低，并且它对于体内DNA复制是必不可少的，大大增强了前导链和滞后链合成的持续合成能力。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其与复制叉的其他蛋白质的相互作用提供了机会。gp2.5的结构将通过单独的gp2.5和与DNA结合的gp2.5的X射线晶体学研究来研究。复合物的结构将直接揭示与DNA的相互作用模式。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-引发酶物理相互作用。这些相互作用对于前导链和滞后链合成的偶联至关重要。gp2.5和T7 DNA聚合酶的相互作用将通过分析性超离心更精确地表征。沉降平衡实验将直接测量这种蛋白质-蛋白质缔合的稳定性和亚基化学计量。该信息将用于制备复合物的结晶。gp2.5和基因4解旋酶引发酶的相互作用将通过X射线晶体学研究进行研究。确定gp2.5-T7 DNA聚合酶全酶复合物或gp2.5-基因4复合物的结构将提供关于复制叉处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.