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BP: Fachverband Biologische Physik
BP 15: Single Molecules
BP 15.3: Talk
Wednesday, February 27, 2008, 14:30–14:45, C 243
Towards resolving single helicase steps on DNA using magnetic tweezers — •Daniel Klaue and Ralf Seidel — Biotechnology Center, Dresden University of Technology, Germany
Replicative helicases drive processive DNA unwinding during DNA replication, the process during which a copy of the genome is synthesized. They are large hexamers, which encircle DNA. ATP hydrolysis in each of the six monomers drives processive movement of the helicase along DNA, which is coupled to DNA unwinding. However, it still remains elusive, how the six ATPase units are coordinated to achieve directional movement. To address this question, we study Large T antigen, a viral replicative helicase, which serves as an important model system for eukaryotic replication. We apply magnetic tweezers in order to follow the DNA unwinding of a single DNA hairpin in real-time. DNA unwinding by T antigen is comparably slow with 1-2 bp s−1. Resolving the bp-sized steps of the helicase along DNA would provide important insight into the coordination of the ATPase units. We therefore tested and improved the resolution limits of the applied magnetic tweezers, where a magnetic microsphere is used to exert force on a single DNA molecule. We achieve sub-nm accuracy in detecting the position of immobilized microspheres. However, we find that DNA bound microspheres can exhibit significant rotational fluctuations thereby limiting the resolution in these experiments. Nonetheless, by carefully selecting the measured microspheres we can obtain nm resolution on a second time scale, which would be sufficient to resolve bp-sized helicase steps.