Probing The Kinetics Of A Model Helicase-Nuclease With A Temperature-Controlled Magnetic Tweezers

Motor protein activities such as ATP hydrolysis and translocation are temperature-dependent. Their characteristic physicochemical parameters, e.g. the ATP coupling efficiency and the translocation step size, can be estimated by studying the kinetics of molecular motors like helicases under different thermal conditions [1]. Here, we present some of the technical developments concerning a temperature-controlled Magnetic Tweezers setup that allows us to perform single-molecule experiments at temperatures in solution of up to 40 °C with a precision of 0.1 °C. Using this instrument we have been able to compare the translocation activity of individual copies of the bacterial DNA helicase-nuclease complex AddAB [2, 3] - an enzyme involved in the initial steps of double-stranded DNA break repair by homologous recombination - at different thermal settings with results obtained from ensemble measurements. Interestingly, although the two complementary approaches give rise to a systematic difference between their corresponding velocities measured at each temperature, they yield almost identical estimates of the kinetic barrier of the translocation process, which turns out to be on the order of 21 kT and hence similar to activation energies observed for other translocating proteins. Experiments to address the coupling between ATP hydrolysis and translocation by AddAB are ongoing and will also be discussed. [1] Seidel, R., et al., “Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I”. EMBO J, 2008. 27(9): p. 1388-98. [2] Yeeles, J.T., et al., “Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease”. Mol Cell, 2011. 42(6): p. 806-816. [3] Carrasco, C., et al., “On the mechanism of recombination hotspot scanning during double-stranded DNA break resection”. Proc Natl Acad Sci USA, 2013. 110(28): p. E2562-71.