SAXS STUDIES ON P1 PARTITION COMPLEXES

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。 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。这一过程被称为分割或分离，是由功能上同源的PAR系统介导的，该系统由一个顺式作用的着丝粒样DNA位点(S)和两个蛋白质ParA和PARB组成。大肠埃希氏菌P1质粒分割仪已被用作分割器的范例。P1para是一个44 kDa的Walker类型的ATPase，它驱动着最后一步的分离。P1PARB是一个38 kDa的DNA结合蛋白，在分离、分配复合体形成和配对的初始步骤中起中介作用。在配位复合体的形成过程中，PARB与大肠杆菌蛋白整合宿主因子(IHF)协同结合到74bp的PARS着丝粒样位点，含有多个A-盒和B-盒。固有弯曲的DNA可以替代IHF，证实它的作用只是将含有Pars臂的A-Box/B-Box结合在一起，从而结合PARB。在初始配位复合体形成后，当多个PARB分子加载到PARS上时，PARB介导了质粒之间的配对。虽然生物化学已经很好地描述了它，但由于缺乏关于分配蛋白及其复合体的结构信息，对分配的详细机制缺乏了解。因此，这项提议的长期目标是将第一阶段平均成绩评估系统作为模型来研究种族隔离的各个步骤。我们的第一个目标是获得初始PARB-IHF-PARS配位络合物的低分辨率结构。为此，我们将利用仅允许结合单个PARB二聚体的PARS位点。随后，我们将包括额外的结合位点来建立更大的分配复合体，使我们能够第一次查看质粒配对，并最终捕获PARB-IHF-PARS(ATP)-PARS预分离复合体。

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.