STRUCTURES OF PROTEIN-DNA COMPLEXES INVOLVED IN EUKARYOTIC REPLICATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在DNA复制发生之前，被称为启动子的特殊AAA+ATPase必须将模板链分开。一种真核型古生菌CDC6/ORc1启动子的单体结构已有报道，但关于起源结合和低聚物形成如何导致启动子的报道很少。最近，我们使用硒蛋氨酸相变技术，解决了一个Cdc6/Orc1异二聚体和基因组起始序列之间的复合体的结构为3.3°的问题。我们希望利用SSRL的同步加速器设施来1)通过重原子导数来改进实验阶段，2)提高这种结构的分辨率，使我们能够在原子水平上深入了解复制启动的控制。初步实验表明，适当的低温保护对分辨率至关重要，我们计划对低温保护剂和渗透强度进行系统筛选，以提高分辨率。一旦复制开始，复制体开始复制基因组，拓扑压力就会在复制分叉之前建立起来。同源二聚体II型拓扑异构酶(TopOII)通过一系列复杂的运动来缓解这种压力，最终导致一条DNA链穿过另一条链。我们最近从酿酒酵母Topo II的晶体中获得了3.2°的天然数据集，该晶体被捕获在与原钒酸链切割反应的中间。我们计划使用SSRL设施来扩展这些数据的分辨率，并从SAD/MAD/SIRAS实验中获得阶段。虽然某些铂盐能产生稳定的衍生物，但对这些晶体的初步实验还没有产生有用的相信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Before DNA replication can take place, specialized AAA+ ATPases known as initiators must separate the template strands. The monomeric structure of one eukaryotic-type archaeal Cdc6/Orc1 initiator has been reported, but very little is known about how origin binding and oligomer formation leads to initiation. We have recently used selenomethionine phasing to solve the structure to 3.3¿ of a complex between a Cdc6/Orc1 hetereodimer and a genomic initiation sequence. We hope to use the synchrotron facilities at SSRL to 1) improve the experimental phases via heavy atom derivatives and 2) improve the resolution of this structure, allowing us to gain insight into the control of replication initiation at an atomic level. Initial experiments have suggested that proper cryoprotection is critical for resolution, and we plan to perform a systematic screen of cryoprotectants and osmotic strengths to improve resolution. Once replication has initiated and the replisome has begun duplicating the genome, topological stress builds ahead of the replication fork. The homodimeric type II topoisomerases (TopoII) relieve this stress through a complex series of movements that culminate in one DNA strand being passed through the other. We have recently obtained a 3.2¿ native dataset from a crystal of the S. cerevisiae TopoII trapped in the middle of a strand cleavage reaction with orthovanadate. We plan to use the SSRL facilities to both extend the resolution of this data and obtain phases from SAD/MAD/SIRAS experiments. Though certain platinum salts yield stable derivatives, initial experiments with these crystals have not yet yielded usable phase information.