Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells

Nat Commun. 2014 Dec 18:5:5820. doi: 10.1038/ncomms6820.

Abstract

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism
  • DNA Repair*
  • DNA Replication*
  • DNA, Bacterial / chemistry*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Microfluidic Analytical Techniques
  • Microscopy, Fluorescence
  • Recombination, Genetic
  • Time-Lapse Imaging

Substances

  • DNA, Bacterial
  • DNA Polymerase III