Tracking enzymatic steps of DNA topoisomerases using single-molecule micromanipulation

Publication Type:

Journal Article

Source:

COMPTES RENDUS PHYSIQUE, EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, Volume 3, Number 5, 23 RUE LINOIS, 75724 PARIS CEDEX 15, FRANCE, p.595-618 (2002)

Keywords:

ATP HYDROLYSIS; DROSOPHILA-MELANOGASTER; ELASTICITY; FORCE; MECHANISM; POSITIVELY SUPERCOILED DNA; RELAXATION; STEADY-STATE ANALYSIS; STRUCTURAL TRANSITIONS; TRANSPORT

Abstract:

In this article, we describe single-molecule assays using magnetic traps and we applied these assays to topoisomerase enzymes which unwind and disentangle DNA molecules. First, the elasticity of single DNA molecule is characterized using the magnetic trap. We show that a twisting constraint may be easily applied and that its effect upon DNA may be measured accurately. Then we describe how the topoisomerase activity may be observed at the single-molecule level giving direct access to the important biological parameters of the enzyme such as velocity and processivity. Furthermore, individual cycles of unwinding can be observed in real time. This permits an accurate characterization of the enzyme's biochemical cycle. The data treatment required to identify and analyze individual topoisomerization cycles will be presented in detail. This analysis is applicable to a wide variety of molecular motors. (C) 2002 Academie des sciences/Editions scientifiques et medicales Elsevier SAS.