Dresden 2014 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 2: DNA/RNA and related enzymes
BP 2.9: Vortrag
Montag, 31. März 2014, 12:00–12:15, ZEU 250
Probing the kinetics of a model helicase-nuclease with a temperature-controlled Magnetic Tweezers — •Benjamin Gollnick1, Carolina Carrasco1, Francesca Zuttion1, Neville S. Gilhooly2, Mark S. Dillingham2, and Fernando Moreno-Herrero1 — 1Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain — 2School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK
Motor protein activities such as ATP hydrolysis and translocation are temperature-dependent; by studying their kinetics under different thermal conditions one can estimate the associated physicochemical parameters. Here, we present 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 - 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.