STRUCTURAL STUDIES ON THE MULTIPROTEIN-DNA, P1 PARTITION COMPLEX

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。 遗传信息的忠实遗传对所有生物体来说都是必不可少的，这需要在细胞分裂时准确的DNA分割(分离)。在原核生物中，分配是由PAR系统介导的，其中以大肠杆菌P1质粒系统为原型，由一个分配位点(S)和两个蛋白(ParA和PARB)组成。为了形成分离所需的分配复合体，P1 PARB必须以某种方式识别位于大的(~74bp)PARS着丝粒中心被IHF急剧弯曲的两端的A-和B-Box DNA基序的复杂排列。这个大型核蛋白复合体的整体结构尚不清楚。有趣的是，尽管不同的PAR系统在其DNA着丝粒位点和PARB蛋白上有所不同，但在所有情况下，PARB蛋白和DNA位点结合形成类似的核蛋白复合体，作为分配机制的成核点。我们最近确定了PARB与PARS-Small结合的结构，这是PARS的右侧部分。这种结构揭示了PARB的关键DNA结合特征，使其能够结合A-盒和B-盒的多种排列。最值得注意的是，该结构还表明PARB作为一个桥梁因子，从而解释了它如何在Pars臂之间结合。然而，为了充分了解分配复合体的形成，需要了解全长P1分配复合体的结构，该结构由PARB、全长PARS和IHF组成。这种复合体的低分辨率结构将提供全长配位复合体的第一个视图，这将标志着在理解大型配分复合体的形成以及它们如何作为分配体机械的成核点方面向前迈出了重要的一步。我们有PARB-IHF-DNA复合体的浓缩液(&gt；10 mg/ml)，目前可供分析。PARB-IHF-DNA复合体纯度高(&gt；95%)。用动态光散射对这些溶液进行了表征，发现它们是单分散的。

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 genetic information, essential for all organisms, requires accurate DNA partition (segregation) at cell division. In prokaryotes, partition is mediated by par systems, for which Escherichia coli P1 plasmid system is a prototype, comprised of a partition-site(s) and two proteins, ParA and ParB. To form the partition complex necessary for segregation, P1 ParB must somehow recognize a complicated arrangement of A- and B-Box DNA-motifs located on opposite ends of a large (~74-bp) parS centromere-site that is sharply bent at its center by IHF. The overall structure of this large, nucleoprotein complex is unknown. Interestingly, although the various par systems differ in their DNA centromere sites and ParB proteins, in all cases, the ParB proteins and DNA sites combine to form similar nucleoprotein complexes that serve as nucleation points for the partition machinery. We recently determined the structure of ParB bound to parS-small, which is the right side portion of parS. This structure reveals key DNA-binding features of ParB that allow it to bind multiple arrangements of A- and B-Boxes. Most remarkably, the structure also shows that ParB acts as a bridging factor, thus explaining how it can bind between parS arms. However, to fully understand partition complex formation requires knowledge of the structure of the full length P1 partition complex, comprised of ParB, full length parS and IHF. A low resolution structure of this complex, which would provide the first view of a full length partition complex, would mark an important step forward in understanding the formation of large partition complexes and how they might serve as a nucleation points for the partition machinery. We have concentrated solutions (> 10 mg/ml) of ParB-IHF-DNA complexes that are currently available for analysis. The ParB-IHF-DNA complex is highly pure (>95% purity). These solutions have been characterized by Dynamic Light Scattering and found to be monodisperse.