The DNA binding properties of the Escherichia coli RecQ helicase.
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.
Dou
Shuo-Xing
SX
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Wang
Peng-Ye
PY
Xu
Hou Qiang
HQ
Xi
Xu Guang
XG
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000495:Allosteric Site; D016880:Anisotropy; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D053484:RecQ Helicases; D012685:Sepharose / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D004247:DNA; D012685:Sepharose; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases