Dresden 2006 – wissenschaftliches Programm
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AKB: Biologische Physik
AKB 6: DNA Mechanics
AKB 6.3: Vortrag
Montag, 27. März 2006, 15:00–15:15, ZEU 260
Tracking of Type I restriction enzymes along DNA — •Ralf Seidel1,2, Joost G. P. Bloom1, Carsten van der Scheer1, Nynke H. Dekker1, Mark D. Szczelkun3, and Cees Dekker1 — 1Kavli Institute of Nanoscience, Delft University of Technology, The Netherlands — 2Biotechnolgisches Zentrum, TU Dresden, Germany — 3Department of Biochemistry, University of Bristol, UK
Type I restriction enzymes are complex cellular machines that cleave foreign, viral DNA using two DNA translocating motor subunits. During the translocation process the core unit of the enzyme complex stays bound to the DNA whilst the motors translocate adjacent DNA and thus pull it towards the enzyme, which results in the formation of large DNA loops. Using magnetic tweezers, we investigated on the level of a single enzyme how the DNA motors track along their DNA template. We found that the motor subunits follow directly the helical pitch of the DNA. Due to the attachment of the motors to the core unit of the enzyme, their rotation around the DNA is inhibited and torsional stress is not released. In this way the DNA gets threaded through the enzyme complex leading to an almost complete untwisting of the DNA in the extruding loop and strongly positively supercoiled DNA in front of the motor. Probing both the translocated distance on the DNA and the amount of generated supercoils, we found that the enzyme translocates 11 ± 2 bp per generated supercoil, which strongly suggests tracking of the DNA helical pitch. We furthermore found that the motors track along the 3’-5’ strand by investigating how small single strand gaps are overcome.