SAXS STUDIES ON P1 PARTITION COMPLEXES

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。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他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的研究机构。 原核遗传物质的忠实遗传需要染色体和质粒在细胞分裂时定向移动和定位到子细胞。 该过程称为分配或分离，由功能同源的par系统介导，par系统由顺式作用的着丝粒样DNA位点和两种蛋白质帕拉和ParB组成。 大肠杆菌P1质粒分配装置已用作分配的范例。 P1帕拉是一个44 kDa的步行者型ATP酶，在最后一步的分配中驱动质粒分离。P1 ParB是一种38 kDa的DNA结合蛋白，介导分离的初始步骤;分区复合物的形成和配对。 ParB和E.大肠杆菌蛋白整合宿主因子（IHF）与含有多个A盒和B盒的parS着丝粒样位点（~74 bp）协同结合形成分配复合物。 内源性弯曲DNA可以替代IHF，证实其作用仅仅是将含有parS臂的A-Box/B-Box结合在一起，从而结合ParB。 在形成初始分配复合物后，ParB介导质粒之间的配对，因为多个ParB分子加载到parS上。虽然它已被生物化学很好地表征，分区的详细机制的理解是缺乏的，在很大程度上，由于缺乏分区蛋白质及其复合物的结构信息。 因此，本提案的长期目标是使用P1标准杆系统作为模型来研究隔离的各个步骤。 我们的第一个目标是获得初始ParB-IHF-parS配分复合物的低分辨率结构。 为此，我们将利用仅允许单个ParB二聚体结合的parS位点。 随后，我们将包括额外的结合位点以构建更大的分区复合物，使我们能够首次观察质粒配对，并最终捕获ParB-IHF-帕拉（ATP）-parS预分离复合物。

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. The faithful inheritance of prokaryotic genetic material requires the directed movement and positioning of chromosomes and plasmids to daughter cells at cell division. This process, called partition or segregation, is mediated by functionally homologous par systems comprised of a cis-acting centromere-like DNA site(s) and two proteins, ParA and ParB. The Escherichia coli P1 plasmid partition apparatus has served as a paradigm for partition. P1 ParA is a 44 kDa Walker type ATPase that drives plasmid separation at the final step of partition. P1 ParB is a 38 kDa DNA-binding protein that mediates the initial steps in segregation; partition complex formation and pairing. In partition complex formation, ParB and the E. coli protein, integration host factor (IHF), bind cooperatively to the ~74 bp parS centromere-like site, which contains multiple A- and B-Boxes, to form the partition complex. Intrinsically bent DNA can substitute for IHF, confirming that its role is simply to bring together the A-Box/B-Box containing parS arms, which bind ParB. After the initial partition complex is formed, ParB mediates pairing between plasmids as multiple ParB molecules load onto parS. Although it has been biochemically well characterized, a detailed mechanistic understanding of partition is lacking due, in large part, to the dearth of structural information on partition proteins and their complexes. Thus, the long terms goals of this proposal are to use the P1 par system as a model to study various steps in segregation. Our first goal is to obtain a low resolution structure of the initial ParB-IHF-parS partition complex. To do this we will utilize a parS site that allows binding of only a single ParB dimer. Subsequently, we will include additional binding sites to build larger partition complexes allowing us to view plasmid pairing for the first time and ultimately to trap a ParB-IHF-ParA(ATP)-parS pre-segregation complex.