Prokaryotic replication

Overview

Bacterial systems form excellent model systems for understanding the intricacies of DNA replication. We study DNA replication in the bacterial system Escherichia coli (E.coli), using two approaches: in vitro single-molecule techniques using isolated, purified components with the aim of gaining direct insight into how individual components of the replication machinery contribute to the efficiency and reliability of DNA replication; and in vivo single-molecule fluorescence microscopy in living E.coli cells to examine the dynamics of replication as they occur within the complexity of the living cell.

Techniques

We employ an array of different techniques depending on the biological questions that we wish to answer. For example, we have used high-throughput single-molecule techniques to the termination of bacterial replication. In this assay, we mechanically unzipped DNA hairpins to mimic the strand separation that normally accompanies DNA replication. In this way we were able to observe how the DNA binding protein Tus blocks the DNA replication machinery when bound to a specific Ter DNA sequence (Figure 1), leading to termination of DNA replication in E.coli. The high-throughput character of these techniques allowed us to achieve high statistics and, in consequence, decipher the scheme underlying the establishment of the Tus-Ter lock.
Tus-Ter lock
Figure 1. Artist’s impression of the magnetic tweezers DNA hairpin experiment, where Tus blocks the force-induced unzipping of a DNA hairpin at a specific Ter DNA site. The Ter DNA acts as a key that fits into a keyhole provided by Tus. (TU Delft – Tremani)

 

 

 

 

 

To follow the activity of the bacterial replisome, which replicates DNA at a rate of approximately 1 kb/s, we convert these instruments into high-speed magnetic tweezers.

In a very different approach, we also use single-molecule fluorescence microscopy techniques to study the dynamics the bacterial replisome in vivo. To make this possible, we make use of different fluorescence proteins that can be tagged to the replisome as well as other proteins involved in replication. The light coming from these fluorescent proteins can be imaged by a very sensitive EMCCD camera. An example of such an image is given below.

xie_data
Figure reference: Yu et al. Probing gene expression in live cells, one protein molecule at a time. Science (2006) vol. 311 (5767) pp. 1600-3

Researchers involved

  • Sam Leachman
  • Sumit Deb Roy
  • Theo van Laar

Current collaborators

  • David Sherratt (Oxford University, UK)
  • Rodrigo Reyes-Lamothe (McGill University, Canada)
  • The Nick Dixon Lab (University of Wollongong, Australia)
  • The Irina Artsimovitch Lab (Ohio State University, USA)

Publications specific to these projects

R. Janissen*, M. Arens*, N. Vtyurina*, B. Eslami-Mossallam, A. Gritsenko, D. de Ridder, I. Artsimovitch, N.H. Dekker, E. A. Abbondanzieri, and A. S. Meyer
Transcriptional regulation is independent of Dps-driven reorganization of DNA
(* = equal contribution)
submitted (2017)

Bojk A. Berghuis, Vlad-Stefan Raducanu, Mohamed M. Elshenawy, Slobodan Jergic, Martin Depken, Nicholas E. Dixon, Samir M. Hamdan, and Nynke H. Dekker
What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments
submitted (2017)

Measuring in vivo protein dynamics throughout the cell cycle using microfluidics
Roy de Leeuw, Peter Brazda, M. Charl Moolman, Belen Solano, and Nynke H. Dekker
in press (2017)

B.A. Berghuis, D. Dulin, Z.-Q. Xu, T. van Laar, B. Cross, R. Janissen, S. Jergic, N.E. Dixon, S.M. Depken, and N.H. Dekker
Strand separation establishes a sustained lock at the Tus–Ter replication fork barrier
Nature Chemical Biology, online publication July 6 (2015) PDF SI news and views

M.C. Moolman*, S. Tiruvadi Krishnan*, J.W.J. Kerssemakers, R. de Leeuw, V. Lorent, D.J. Sherratt, and N.H. Dekker
The progression of replication forks at natural replication barriers in live bacteria
Nucleic Acids Research, online publication May 10 (2016) PDF SI
(* = equal contribution)

B.A. Berghuis, M. Koeber, T. van Laar, and N.H. Dekker
High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers
Methods, online publication March 30 (2016) PDF

M. Charl Moolman*, Jacob W.J. Kerssemakers*, and Nynke H. Dekker
Quantitative analysis of intracellular fluorescent foci in live bacteria
Biophysical Journal, online publication September 4 (2015)
(* = equal contribution) PDF SI

S. Tiruvadi Krishnan, M. C. Moolman, T. van Laar, A. S. Meyer, and N.H. Dekker
Essential validation methods for E. coli strains created by chromosomal engineering
Journal of Biological Engineering, published July 1 (2015) PDF

M. Charl Moolman, Sriram Tiruvadi Krishnan, Jacob W.J. Kerssemakers, Aafke van den Berg, Pawel Tulinski, S. Martin Depken, Rodrigo Reyes-Lamothe, David J. Sherratt, and Nynke H. Dekker
Slow unloading leads to DNA-bound beta-2 sliding clamp accumulation in live Escherichia coli cells
Nature Communications, online publication Dec. 18 (2014) PDF SI

M.C. Moolman, Z. Huang, S. Tiruvadi Krishnan, J.W.J. Kerssemakers and N.H. Dekker
Electron-beam Fabrication of a Microfluidics Device for Submicron-scale Bacteria
J. Nanobiotechnology, published online April (2013) PDF

Brian P. English, Vasili Hauryliuk, Arash Sanamrad, Stoyan Tankov, Nynke H. Dekker, and Johan Elf
Single Molecule Investigations of the Stringent Response Machinery in Individual Living Bacterial Cells
Proceedings of the National Academy of Sciences USA (2011) PDF SI