Dresden 2011 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 4: DNA \& DNA Enzymes
BP 4.7: Vortrag
Montag, 14. März 2011, 16:00–16:15, ZEU 260
Type III restriction enzymes use 1D diffusion to communicate the relative orientation of their distant target sites — •Friedrich W. Schwarz1, Julia Tóth2, Kara van Aelst2, Mark D. Szczelkun2, and Ralf Seidel1 — 1BIOTEC TU-Dresden — 2University of Bristol, UK
Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. The communication process is strictly dependent on ATP hydrolysis catalyzed by their superfamily 2 helicase domains. Given the similarity to Type I restriction enzymes, which couple ATP hydrolysis to directed motion on DNA, unidirectional loop translocation has been the suggested communication mechanism for Type III enzymes. Based on magnetic tweezers single-molecule cleavage experiments and ATPase measurements we suggest an alternative inter-site communication mechanism using 1D diffusion along the DNA contour. In order to verify this hypothesis we directly visualized the motion of Q-dot labeled Type III restriction enzymes along DNA. For this we used a setup that combines magnetic tweezers with total internal reflection fluorescence microscopy. The enzymes undergo a fast diffusive motion along DNA, capable of scanning kb distances per second. We also found that the affinity of the enzymes to non-specific and specific DNA is regulated by the presence of ATP, suggesting that ATP hydrolysis acts as a trigger for diffusion. Thus Type III restriction enzymes are the first DNA-modifying enzymes which communicate target site orientations over long distances via 1D diffusion.