Behavior of Tn3 resolvase in solution and its interaction with res.
The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
Marcelo
M
Nöllmann
Olwyn
O
Byron
W Marshall
WM
Stark
D016880:Anisotropy; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001703:Biophysics / Q000379:methods*; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009502:Neutrons; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013696:Temperature; D045564:Transposon Resolvases / Q000737:chemistry*; D014461:Ultracentrifugation; D014965:X-Rays
D009841:Oligonucleotides; D004247:DNA; C473491:Tn3 resolvase; D045564:Transposon Resolvases; C473494:gamma delta resolvase