Nucleic Acids Research Advance Access originally published online on February 14, 2008
Nucleic Acids Research 2008 36(6):1976-1989; doi:10.1093/nar/gkm1174
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2008, Vol. 36, No. 6 1976-1989
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding
1Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, 2School of Life Science, East China Normal University, Science Bld., 3663 North Zhongshan Rd., Shanghai 200062, China and 3CNRS, UMR 2027, Institut Curie – Section de Recherche, Centre Universitaire, Bâtiment 110, F-91405 Orsay, France
*To whom correspondence should be addressed. Tel: +86 10 8264 9568; Fax: +86 10 8264 0224; Email: sxdou{at}aphy.iphy.ac.cn
Correspondence may also be addressed to Xu Guang Xi. Tel: +33 1 6986 3181; Fax: +33 1 6986 9429; Email: xgxi{at}curie.u-psud.fr
Received November 5, 2007. Revised December 20, 2007. Accepted December 21, 2007.
PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3'-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of 
2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of 9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed.