Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Single-molecule methods to probe the coordination of E. coli DNA replication (#301)

Richard Spinks 1 , Nick Dixon 1 , Antoine van Oijen 1
  1. School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia

The fundamental asymmetry of DNA replication and has long implied the existence of a refined set of coordination rules that govern the replisome. However, recent single-molecule observations of DNA replication have revealed more intrinsic stochasticity in replisome function, and thus question the necessity of a coordinated replisome.

This work aims to reconcile previous DNA replication models by directly detecting the presence or absence of coordination mechanisms within the E. coli replisome. By marrying protein biochemistry, microfluidics and single-molecule fluorescence techniques, we are able to determine the relative movements of the DnaB helicase and the Polymerase III holoenzyme during reconstituted DNA replication events, and thus infer their coordinated or independent action.