This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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