Single Molecule Studies of DNA-Binding Proteins: Development of New Covalent DNA Anchoring Techniques for the Study of Rupture Forces of Replication Blocks

Abstract

Magnetic Tweezers (MT) are a powerful technique to investigate the dynamics and kinetics of biomechanical processes in-vitro on a single-molecule level. In certain cases, it is necessary to apply unusually high pulling forces for the mechanochemical characterization of biomolecule structures and complexes, such as protein-nucleic acid complexes, DNA and RNA conformational overstretching conformation transition phenomena, or the unfolding of polymers and proteins at high resolution.To achieve high pulling forces using MT, we have redesigned the anchoring strategy of biomolecules to the surface and magnetic bead, employing a bottom-up covalent chemisorption procedure. The development covers several aspects such as surface passivation to avoid aspecific physisorption, flexible and interchangeable covalent binding strategies using inert poly(ethylene glycol) linkers, and the characterization of chemical stability of tethered dsDNA sample constructs and overall coating density. Using this novel approach, dsDNA constructs have reproducibly and reliably withstood pulling forces of > 140 pN over long observation times.We have applied our novel anchoring strategy to anchor DNA hairpins containing a single replication termination (ter) sequence. In E.coli, the DNA binding protein Tus binds to ter sites and is known to bring approaching replication forks to a halt by blocking strand separation. Upon opening our DNA hairpin in the presence of counter-helicase Tus, we mimicked replisome activity, and strand separation was demonstrably blocked. However, the barrier imposed was larger than our non-covalent anchoring method could withstand; it was impossible to ‘break the lock’. Employing our new method, we are able to apply forces > 140 pN to the Tus:ter complex, allowing us to characterize the energy landscape of this system.