After DNA replication, two daughter copies of bacterial chromosomes or low copy number plasmids must be segregated into two daughter cells in order to avoid rapid chromosome loss. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. One class of such systems involve a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of centromere, and two protein factors, one binds to the centromere and the other an ATPase with non-specific DNA binding activity. E. coli P1 plasmid and F plasmid are both equipped with such systems. The centromere of P1 plasmid is called ParS, to which ParB protein binds, and ParA is the ATPase. The centromere of F plasmid is called SopC, to which SopB protein binds, and SopA is the ATPase. In vivo imaging studies on some of these systems have demonstrated oscillating focus formation of the ATPase protein and accompanied oscillation of the plasmid DNA within the cell prior to DNA replication. After replication, one DNA copy stays near one end of the cell and the other copy moves toward the other end prior to cell division. However, detailed molecular mechanisms of these bio-molecular transport reaction systems are still poorly understood, due in part to the absence of suitable cell free reaction system to detect the DNA movements. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques. Techniques and instruments have been developed to study these reactions at the single molecule detection level by using a sensitive fluorescence microscope/CCD camera system. Using GFP-tagged ParA and fluorescent dye-coupled ParB proteins, association/dissociation dynamics of these proteins with DNA molecules immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: ParA, in the presence of ATP associates with non-specific DNA with rapid on- and off-rates. No other nucleotide analogues have been found to be able to substitute ATP for ParA-DNA association. ParA conformational change induced by ATP binding has been observed. Pre-steady state kinetic analysis of the ParA ATPase reaction and the ATP-induced conformational change of ParA have been studied. The ATP-induced ParA conformational change, which could take place prior to ATP hydrolysis is required for ParA DNA binding. These observations will be combined to formulate a model for the mechanism of action of this reaction system. We also are in the process of initiating a parallel study of the F plasmid partitioning reaction. Fluorescence-labeled SopA and SopB proteins have been constructed and purified in active forms. The reaction system studied here is an example of a novel biomolecular transport reaction, and the experimental techniques developed here will be exploited for the parallel studies of mechanistically related reaction systems.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$312,826
Indirect Cost
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State
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Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2017) Brownian ratchet mechanisms of ParA-mediated partitioning. Plasmid 92:12-16
Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2017) Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids. Biophys J 112:1489-1502
Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2015) Directed and persistent movement arises from mechanochemistry of the ParA/ParB system. Proc Natl Acad Sci U S A 112:E7055-64
Vecchiarelli, Anthony G; Taylor, James A; Mizuuchi, Kiyoshi (2015) Reconstituting ParA/ParB-mediated transport of DNA cargo. Methods Cell Biol 128:243-69
Vecchiarelli, Anthony G; Seol, Yeonee; Neuman, Keir C et al. (2014) A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force. Bioarchitecture 4:154-9
Vecchiarelli, Anthony G; Neuman, Keir C; Mizuuchi, Kiyoshi (2014) A propagating ATPase gradient drives transport of surface-confined cellular cargo. Proc Natl Acad Sci U S A 111:4880-5
Hwang, Ling Chin; Vecchiarelli, Anthony G; Han, Yong-Woon et al. (2013) ParA-mediated plasmid partition driven by protein pattern self-organization. EMBO J 32:1238-49
Vecchiarelli, Anthony G; Hwang, Ling Chin; Mizuuchi, Kiyoshi (2013) Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proc Natl Acad Sci U S A 110:E1390-7
Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Funnell, Barbara E (2012) Surfing biological surfaces: exploiting the nucleoid for partition and transport in bacteria. Mol Microbiol 86:513-23
Vecchiarelli, Anthony G; Han, Yong-Woon; Tan, Xin et al. (2010) ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition. Mol Microbiol 78:78-91