J-F. Allemand, G.Charvin, T.Lionnet, M.-N. Dessinges, D. Bensimon and V.Croquette
Laboratoire de Physique Statistique, ENS, 24 rue Lhomond, Paris 75005
The recent developments of single molecule manipulation tools as opened a new vista on the study of biological processes at their most fundamental level. We have developed an original method to manipulate single molecule: the magnetic trap. It allows one to twist and stretch single DNA molecule from 0.005 to 100 pN. Our technique allows for a detailed analysis of the enzymatic cycle of various molecular motors. In particular, one gains access to the probability distribution of enzymatic rates and their stress-dependence. In the past few years we have used that technique to study a number of enzymes whose interaction with DNA is of prime importance: DNA-polymerase, topoisomerases, helicases, regulation factors, etc. We have been able to monitor in real time the activity of these proteins and measure their rates as a function of force, ATP concentration, ionic conditions, etc. These studies have shed a new light on the function of these enzymes. In particular they have shown that their activity and processivity is many times larger than previously estimated from bulk data.
Helicases.
By monitoring in real time the change in extension of a DNA molecule as it is unzipped by a single helicase (see Fig.1) we were able to measure its rate of unwinding (~250 bps/s), processivity (~400 bps), on-time (~1.8 sec) and step-size (~5 bps). We also observed a totally unexpected strand-switching of the helicase, were the enzyme unzipping the DNA by moving 3’-5’ on one strand switches to the other anti-parallel strand and (moving 3’-5’ on that strand) allows the ATP-dependent slow rewinding of the two DNA strands (see Fig.1).
Topoisomerases.
We have studied the unbraiding of two DNA molecule by a single Eukaryotic or Prokaryotic topoisomerase. This configuration which mimics the intertwining of the chromosome strands during replication allows us to address the role played by topoisomerases during replication. Central to this issue is the understanding of the decatanation activity of the prokaryotic enzyme, which substrate during replication (right-handed braids) presented a DNA crossing geometry that was shown to be unfavorable for enzymatic activity (similar to negative supercoils that the enzyme relaxes very poorly). In fact to our surprise, we were able to observe a normal unbraiding activity of the Prokaryotic enzyme on these right-handed braids when they were highly intertwined, but none at low braiding. We have shown that this activity results from the formation (at high braidings) of supercoils of right-handed braids that present left-handed DNA crossings that are undone by the enzyme, see Fig.2.
Ref.: "Single molecule study of DNA unlinking by eukaryotic and prokaryotic type II topoisomerases", G. Charvin, D. Bensimon and V. Croquette, Proc.Nat.Acad.Sci. USA 100, 9820-9825 (2003).
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