PMID:14665634

Citation	No author listed (2004) The DNA binding properties of the Escherichia coli RecQ helicase. J. Biol. Chem. 279:6354-63
Abstract	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'-(beta,gamma-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 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=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.
Links	PubMed Online version:10.1074/jbc.M311272200
Keywords	Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Allosteric Site; Anisotropy; Binding Sites; Binding, Competitive; DNA/chemistry; DNA Helicases/chemistry; DNA Helicases/metabolism; DNA, Single-Stranded/chemistry; DNA-Binding Proteins/chemistry; Dose-Response Relationship, Drug; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; Oligonucleotides/chemistry; Protein Binding; RecQ Helicases; Sepharose/chemistry; Temperature; Thermodynamics; Time Factors
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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
recQ:Gene_Product(s)		GO:0003677	DNA binding	IDA: Inferred from Direct Assay		F	The interaction of RecQ with DNA was shown using gel mobility shift assays (Figure 2).	complete
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