Probing DNA Helicase Kinetics with Temperature-Controlled Magnetic Tweezers

Benjamin Gollnick, Carolina Carrasco, Francesca Zuttion, Neville S. Gilhooly, Mark S. Dillingham, Fernando Moreno-Herrero*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

21 Citations (Scopus)
1 Downloads (Pure)


Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 kBT) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.

Original languageEnglish
Pages (from-to)1273-1284
Number of pages12
Issue number11
Early online date14 Nov 2014
Publication statusPublished - 18 Mar 2015


  • activation energy
  • AddAB helicase-nuclease
  • magnetic tweezers
  • single-molecule studies
  • temperature control


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