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M311272200v1
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Papers In Press, published online ahead of print December 9, 2003
J. Biol. Chem, 10.1074/jbc.M311272200
Submitted on October 14, 2003
Revised on December 7, 2003
Accepted on December 9, 2003

The DNA binding properties of the Escherichia. coli RecQ helicase

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

Biology, Ecole normale superieure de Cachan, cachan, Cachan 94235

Corresponding Author: xi{at}lbpa.ens-cachan.fr

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. In order to determine the thermodynamic parameters for the interaction of E. 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 one protein monomer per 10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP or ADP. Analyses of RecQ helicase’s 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 × 106 M-1, and a cooperativity parameter of  = 25.5 ± 1.2. Both ssDNA and dsDNA 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 ssDNA in the presence of 1.5 mM AMPPNP, while both AMPPNP and ADP had no detectable effect on dsDNA binding over a large range of nucleotide cofactor concentrations.


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