After DNA replication, two daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure inheritance. 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 three components;a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a centromere, and two protein factors, one binds to the centromere and the other an ATPase with ATP-dependent 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, the detailed molecular mechanism of these bio-molecular transport reaction systems is still poorly understood. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of these reaction systems by combining a variety of techniques, including exploitation of cell-free reaction systems we recently established that recapitulates aspects of the in vivo system dynamics. Techniques and instruments have been developed to study these dynamic reaction systems by using a sensitive fluorescence microscope/CCD camera system. By using fluorescence-labeled ParA and ParB proteins, or SopA and SopB proteins, association/dissociation dynamics of these proteins with DNA molecules immobilized on a slide glass surface were monitored under a variety of reaction conditions. We learned that ParA, or SopA, in the presence of ATP, associates with non-specific DNA with rapid on- and off-rates. A pre-steady state kinetic analysis of the ParA ATPase reaction and the ATP-induced conformational change of ParA have also been studied. The ParA conformational change necessary for DNA binding has been observed to take place with a time delay following ATP binding, leading to a mechanistic model of plasmid DNA motion. We have successfully reconstituted cell-free systems to observe ATP-driven dynamic behaviors of the fluorescence-labeled plasmid DNA carrying parS, or sopC, in the presence of ParA and ParB proteins, or SopA and SopB proteins, within a flow cell coated by non-specific DNA. Mechanistic details of the ATP-driven plasmid DNA dynamics are currently studied.
|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|
|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|
|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; 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|