Viral replication

Overview

Viral systems for excellent model systems for understanding the intricrdrp_replication_smaller_50_then_25acies of DNA and RNA replication. To this effect, we study RNA synthesis in viruses, focusing on the mechanisms employed by RNA-dependent RNA polymerases (figure at right). This has led to an understanding of the tendency of these polymerases to make errors during the synthesis process. This tendency towards misincorporation can be influenced by the presence of nucleotide analogs and, as such, provide ways of developing new antivirals.

Technique

We use high-throughput single-molecule techniques to examine nucleotide synthesis by viral polymerases. The high-throughput character of these techniques allows us to achieve high statistics and, in consequence, test quantitative models for polymerase mechanisms. Our assays consist of tracking tethered RNA molecules as they are converted from double-stranded RNA to single-stranded RNA by a single RNA-dependent RNA polymerase. Due to the applied force on the bead, and the fact that single-stranded DNA is more extensible than the double-stranded helix, we see this as a length change over time. This can then be converted into a number of RNA nucleotides synthesised over time (figure below).

dwelltime

Using magnetic tweezers we track a single enzyme as it polymerizes RNA over time. The dynamics of this process allows us to gain insight into the energetics and kinetics involved in RNA synthesis.

Researchers currently involved

  • Richard Janissen
  • Theo van Laar

Current collaborators

  • Craig Cameron (Penn State University)
  • Shin-Ru Shih (Chang Gung University, Taiwan)
  • Martin Depken (TU Delft)

Publications specific to these projects

David Dulin, Jamie J. Arnold, Theo van Laar, Hyung-Suk Oh, Cheri Lee, Daniel A. Harki, Martin Depken, Craig E. Cameron, and Nynke H. Dekker
Incorporation of nucleotide analogues by an RNA-dependent RNA polymerase leaves fingerprints in the pausing kinetics
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)

D. Dulin, Z. Yu, T. Cui, B.A. Berghuis, S.M. Depken, and N.H. Dekker,
Real-time observation of replicative helicase assembly onto single-stranded DNA
submitted (2017)

D. Dulin, I.D. Vilfan, B. Berghuis, M. Poranen, M. Depken, and N.H. Dekker
Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site
Nucleic Acids Research, online publication October 22 (2015) PDF

D. Dulin, B.A. Berghuis, M. Depken, and N.H. Dekker
Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments
Current Opinion in Structural Biology, online publication October 1 (2015) PDF

D. Dulin, I.D. Vilfan, B.A. Berghuis, S. Hage, D. Bamford, M. Poranen, S.M. Depken, and N.H. Dekker
Elongation-competent pauses govern the fidelity of a viral RNA-dependent RNA polymerase
Cell Reports, online publication Feb. 12 (2015) PDF Extended PDF

D. Dulin, I.D. Vilfan, B. Berghuis, M. Poranen, M. Depken, and N.H. Dekker
Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site
Nucleic Acids Research, online publication October 22 (2015) PDF

Jan Lipfert, Gary M. Skinner, Johannes M. Keegstra, Toivo Hensgens, Tessa Jager, David Dulin, Mariana Koeber, Zhongbo Yu, Serge P. Donkers, Fang-Chieh Chou, Rhiju Das, and Nynke H. Dekker
Double-Stranded RNA under Force and Torque: Similarities and Striking Differences to Double-Stranded DNA
Proceedings of the National Academy of Sciences (USA) 111 (43), 15408–15413 (2014) PDF SI

I.D. Vilfan, A. Candelli, S. Hage, A.P. Aalto, M.M. Poranen, D.H. Bamford and N.H. Dekker
Reinitiated Viral RNA-dependent RNA Polymerase Resumes Replication at a Reduced Rate
Nucleic Acids Research, doi:10.1093/nar/gkn836, (2008) PDF SI