Crystallographic Investigations of Replication Factor C

描述（由申请人提供）：细菌和真核生物DNA复制和修复的一个重要组成部分是所谓的钳加载器，一个五个亚基复合物，利用水解的能量在DNA周围组装二聚体或三聚体环形环（钳）。本文提出的研究计划包括：1)测定来自S. cerevisiae的真核clamp loader复合物，复制因子C （RFC）在“开放”（atp结合）和“封闭”（无核苷酸）构象中的晶体结构；2)测定RFC:PCNA:DNA复合物在水解事件发生前后的晶体结构。所有五个酵母RFC亚基都可以在大肠杆菌中共表达，并纯化到均匀性，数量足以进行结晶。这些结构将为夹具加载周期的关键步骤提供大量的结构信息。酵母RFC复合物的结构对于建立密切相关的人类RFC复合物的模型非常重要，而对原核和真核复合物的比较将扩大我们对箝位加载器基本特性的理解。

DESCRIPTION (provided by applicant): An essential component of bacterial and eukaryotic DNA replication and repair is the so-called clamp loader, a five subunit complex that assembles dimeric or trimeric toroidal rings (clamps) around DNA using the energy of hydrolysis. The research plan presented here proposes 1) crystal structure determination of the eukaryotic clamp loader complex from S. cerevisiae, Replication Factor C (RFC), in the 'open' (ATP-bound) and "closed' (no nucleotide) conformations, and 2) crystal structure determination of a RFC:PCNA:DNA complex before and after a hydrolysis event has occurred. All five yeast RFC subunits can be coexpressed in E. coli and purified to homogeneity in quantities sufficient for crystallization. These structures will offer a tremendous amount of structural information for critical steps in the clamp loading cycle. Structures of the yeast RFC complex will be important for modeling the closely related human RFC complex, and comparison of prokaryotic and eukaryotic complexes should expand our understanding of fundamental properties governing clamp loaders.