SECONDARY AND TERTIARY STRUCTURAL-CHANGES IN GAMMA-DELTA RESOLVASE - COMPARISON OF THE WILD-TYPE ENZYME, THE I110R MUTANT, AND THE C-TERMINAL DNA-BINDING DOMAIN IN SOLUTION

gamma delta Resolvase is a site-specific DNA recombinase (M-r 20.5 kDa ) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and ter tiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were f ound to destabilize the tertiary structure of the DBD dramatically, wi th concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold a ppears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of t he three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three h elices of the DBD structure and changes in the linker segment in compa rison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catal ytic core of similar to 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occu rs on binding DNA. These results support a subunit rotation model of s trand exchange that involves the partial unfolding of the catalytic do mains.