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

M. Charl Moolman,Martin Depken,Rodrigo Reyes-Lamothe,Jacob W. J. Kerssemakers,Pawel Tulinski,David J. Sherratt,Aafke van den Berg,Nynke H. Dekker,Sriram Tiruvadi Krishnan

doi:10.1038/ncomms6820

M. Charl Moolman, Martin Depken + Show 7 more

Open Access

https://doi.org/10.1038/ncomms6820

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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.

Highlights

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair
The in vivo dynamics of b2-clamps measured in single cells
By using in vivo single-molecule fluorescence microscopy in combination with microfluidics, we were able to investigate the detailed dynamics of an essential component of the replisome, the b2-clamp, during DNA replication in live E. coli cells

Summary

Introduction

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. The clamp-loader is thought to chaperone DNA Pol III onto a newly loaded b2-clamp[23] and to unload inactive DNA-bound b2clamps via the d-subunit[24] During all of these reactions, the loader complex and the various clamp binding proteins compete for the carboxy (C)-terminal face of the clamp. While leading-strand replication is thought to be continuous, utilizing only a single b2-clamp, the lagging-stand template is copied in discrete 1–2 kb Okazaki fragments[28], each utilizing a separate b2-clamp These fragments are initiated by the continuous formation of 10–12 nt RNA primers by the primase (DnaG), which, together with the helicase (DnaB), sets the replication fork clock[29]. Since the number of Okazaki fragments (2,000–4,000) for the 4.6 Mbp genome is roughly an order of magnitude greater than the average number of b2-clamps per cell in a nutrient-rich culture[24,30], continuous recycling of b2-clamps is necessary for total genome replication to occur

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