An Isaac Newton Institute Workshop

Towards a Predictive Biology

Single molecule study of DNA/protein interactions

20th January 2004

Author: V. Croquette (CNRS)

Abstract

We have developed a magnetic tweezers setup that allows us to stretch and to twist one or two DNA molecules, and so to control the extension, the supercoiling or the braiding state of DNA molecules. This single DNA micromanipulation experiment permits one to monitor in real-time the activity of DNA processing enzymes through the nanometer scale change of extensions that they produced. In particular, the replication of DNA involves enzymes such as Helicases which separate the DNA strands, polymerases which replicate them and Topoisomerases which disentangle and relax the torsion in the molecule during this process. The understanding of the molecular mechanisms involved in those processes is far from perfect and the kinetics is mostly known from bulk experiments where it is hindered by the diffusion step required by the enzyme to find its substrate.

We shall describe our measurements of the kinetic properties of a few enzymes among helicases, topoisomerases, transcription factors and chromatin remodeling factor. We have evidence that those enzymes are active during bursts, presenting a well defined velocity but random duration and processivity.