STRUCTURAL STUDIES ON THE MULTIPROTEIN-DNA, P1 PARTITION COMPLEX

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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.