STRUCTURES OF PROTEIN-DNA COMPLEXES INVOLVED IN EUKARYOTIC REPLICATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在DNA复制发生之前，被称为启动子的特殊AAA+ ATP酶必须分离模板链。一个真核类型的古细菌Cdc 6/Orc 1启动子的单体结构已被报道，但很少有人知道如何起源绑定和寡聚体的形成导致启动。我们最近使用硒代甲硫氨酸定相来解决Cdc 6/Orc 1异二聚体和基因组起始序列之间的复合物的结构。我们希望使用SSRL的同步加速器设施来1）通过重原子衍生物改善实验阶段，2）提高这种结构的分辨率，使我们能够深入了解原子水平上复制起始的控制。初步实验表明，适当的冷冻保护是至关重要的分辨率，我们计划进行系统的筛选冷冻保护剂和渗透强度，以提高分辨率。一旦复制开始，复制体开始复制基因组，拓扑压力在复制叉之前建立。同二聚体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链通过另一条DNA链。我们最近从S.在与原钒酸盐的链切割反应中间捕获的酿酒酵母TopoII。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实验中获得相位。虽然某些铂盐产生稳定的衍生物，但这些晶体的初步实验尚未产生可用的相位信息。

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.