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J. Biol. Chem., Vol. 279, Issue 8, 6354-6363, February 20, 2004

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The DNA Binding Properties of the Escherichia coli RecQ Helicase^*

Shuo-Xing Dou, Peng-Ye Wang, Hou Qiang Xu, and Xu Guang Xi¶

From the Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China and Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113, Ecole Normale Supérieure de Cachan, 61 avenue du Président Wilson, 94235 Cachan cedex, France

The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(,-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 °C of K = 6.7 ± 0.95 x 10⁶ M^-1 and a cooperativity parameter of = 25.5 ± 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3–4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.

Received for publication, October 14, 2003 , and in revised form, December 7, 2003.

^* This work was supported by the CNRS (to X. G. X.), the National Natural Science Foundation of China Grants 60025516 and 10334100, and the Innovation Project of the Chinese Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 33-1-47-40-68-92; Fax: 33-1-47-40-76-71; E-mail: xi{at}lbpa.ens-cachan.fr.

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