Dresden 2009 – scientific programme

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BP: Fachverband Biologische Physik

BP 7: Poster I

BP 7.49: Poster

Monday, March 23, 2009, 17:45–20:00, P3

The unwinding mechanism of the hexameric helicase Large Tumor Antigen — •Daniel Klaue and Ralf Seidel — Biotechnology Center, TU Dresden, Germany

Helicases are ATP-driven molecular motors that processively unwind dsDNA by shearing apart the individual strands . The mechanisms by which helicases accomplish strand separation are heavily debated. Two extreme possibilities are either a passive mechanism, in which reannealing of stochastically opened base pairs at the unwinding junction is sterically prevented , or an active mechanism in which the helicase actively ruptures base pairing. Whereas for the latter case the helicase velocity should be force independent, for the first case a strong force dependence is expected. Recently for hexameric helicases from bacteriophages, a largely passive DNA unwinding mechanism has been found. Here we investigate the eukaryotic hexameric helicase Large Tumor Antigen (T-antigen) from Simian Virus 40 on the level of a single molecule using magnetic tweezers, where unwinding of a DNA hairpin can be observed in real time. In contrast to its prokaryotic counter parts we find that within error DNA unwinding by T-antigen is force independent in agreement with an active unwinding mechanism. Interestingly, the refolding of the DNA, when T-Antigen passes the center of the hairpin and translocates on the single strand, occurs faster than unwinding. This suggests that the active unwinding occurs ahead of the unwinding junction which is shielded against applied force. In agreement with an active unwinding mechanism we also find that T-antigen is one of the most processive helicases known so far.