Dynamic self-organization of bacterial DNA segregation machinery in cell-free reaction

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NIDDK	BIOPHY-5

* FARE Award Winner

Authors

• LC Hwang
• YW Han
• AG Vecchiarelli
• BE Funnell
• K Mizuuchi

Abstract

DNA segregation is a vital process that ensures the faithful transmission of genetic material during cell division. Eukaryotes use the polymerization/depolymerization of tubulin to separate chromosomes. In bacteria, plasmids have evolved segregation machineries to partition replicated DNA to the daughter cells. P1 phage lysogenizes as a low-copy-number plasmid in Escherichia coli. It encodes only three components for this reaction: a parS sequence acting as the partition site and two proteins, ParA and ParB. Previous studies showed ParB binds to parS, forming a partition complex. ParA, an ATPase and an ATP-dependent DNA-binding protein is thought to couple the ATP hydrolysis energy to drive plasmid motion. However, how these system components work together to drive plasmid segregation remains unclear. To elucidate the mechanism of P1 plasmid partition, we reconstituted the partition system in vitro and visualized protein dynamics with total-internal-reflection-fluorescence microscopy. We coated a flow cell surface with non-specific DNA to mimic the nucleoid surface. ParA-GFP, ParB, parS-Alexa647 plasmid and ATP were flowed into the flow cell. The partition complexes containing ParA, ParB and parS anchored themselves to the protein-coated DNA surface and displayed dynamics analogous to in vivo studies. We propose a diffusion-ratchet model for plasmid segregation based on our results.

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