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. Pre-steady state kinetic analysis of the ParA ATPase reaction has revealed presence of a slow conformational change of ParA upon ATP binding prior to ATP hydrolysis. Direct kinetic analysis of the conformational change is currently in progress. 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.
