The survival of any species demands the faithful inheritance of genetic information. Essential to this process are the directed movements and positioning of chromosomes or plasmids such that they are accurately distributed to the daughter cells at cell division. Although prokaryotes do not undergo the complex mitotic steps associated with eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or par systems. Coordination of this process requires precise protein-DNA and protein-protein interactions carried out by par systems. All known plasmid encoded par loci specify three components: a cis-acting centromere site (parS), and two proteins, ParB and ParA. In the E. coli P1 par system, which has served as a paradigm for understanding partition, the genes for ParA and ParB form an operon, and the approximately 74 bp partition site, parS, is located immediately downstream of parB . ParB, a 38 kDa protein with no sequence homology to any protein, is a DNA-binding protein and, along with IMF or alone, binds to a highly complex centromere-like site, parS, to form the partition complex. ParA, a 44 kDa Walker-type ATPase, utilizes the energy of ATP hydrolysis to drive plasmid separation after interacting with ParB in the partition complex. Our recent structure determinations of ParB and the minimal partition site have revealed novel DNA-binding characteristics of ParB that explain its ability to bind, spread and pair plasmids. Thus, these structures have provided unprecedented insight into the mechanism of partition complex formation. Completely unknown, however, are structural bases for plasmid separation, the step carried out by ParA. Thus, in this proposal we will build on our recent progress towards a full elucidation of P1 partition with the following Specific Aims: (1) fully elucidate the mechanism of P1 partition complex formation through structural and biochemical studies on the ParB-parS partition complex. (2) Clarify the mechanism of P1 plasmid separation via structural and biochemical studies on the key end states of P1 ParA (apoParA, ParA-ADP and the ParA(K188Q)-(AMP-PCP)-ParB(1-28) complex). These structures will provide the foundation for understanding how partition systems function to drive chromosome segregation in prokaryotes and may provide potential points of therapeutic intervention against pathogenic bacteria, which also depend on par systems for segregation and thus, survival. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-IDM-H (03))
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Dearolf, Charles R
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University of Texas MD Anderson Cancer Center
Other Domestic Higher Education
United States
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Schumacher, Maria A; Zeng, Wenjie; Huang, Kuo-Hsiang et al. (2016) Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein, ZapC. J Biol Chem 291:2485-98
Schumacher, Maria A; Tonthat, Nam K; Lee, Jeehyun et al. (2015) Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages. Science 349:1120-4
Hover, Bradley M; Tonthat, Nam K; Schumacher, Maria A et al. (2015) Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis. Proc Natl Acad Sci U S A 112:6347-52
Schumacher, Maria A; Tonthat, Nam K; Kwong, Stephen M et al. (2014) Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein. Proc Natl Acad Sci U S A 111:9121-6
Murray, David S; Chinnam, Nagababu; Tonthat, Nam Ky et al. (2013) Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism. J Biol Chem 288:35801-11
Tonthat, Nam K; Milam, Sara L; Chinnam, Nagababu et al. (2013) SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid. Proc Natl Acad Sci U S A 110:10586-91
Schumacher, Maria A; Chinnam, Nagababu; Ohashi, Tomoo et al. (2013) The structure of irisin reveals a novel intersubunit ?-sheet fibronectin type III (FNIII) dimer: implications for receptor activation. J Biol Chem 288:33738-44
Dobruk-Serkowska, Aneta; Caccamo, Marisa; Rodríguez-Castañeda, Fernando et al. (2012) Uncoupling of nucleotide hydrolysis and polymerization in the ParA protein superfamily disrupts DNA segregation dynamics. J Biol Chem 287:42545-53
Dupaigne, Pauline; Tonthat, Nam K; Espéli, Olivier et al. (2012) Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome. Mol Cell 48:560-71
Schumacher, Maria A (2012) Bacterial plasmid partition machinery: a minimalist approach to survival. Curr Opin Struct Biol 22:72-9

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