STUDIES OF THE DIMERIZATION AND DOMAIN-STRUCTURE OF GAMMA-DELTA RESOLVASE

Gammadelta Resolvase is a site-specific recombinase (20.5 kDa) that ca talyzes the resolution of a negatively supercoiled plasmid to a catena ted pair of circular DNA products (Reed, R. R. (1981) Cell 25, 713-719 ). Cross-linking experiments and size exclusion high pressure liquid c hromatography analysis of isolated fragments corresponding to specific proteolytic cleavage indicate that the intact enzyme and the large fr agment exist in a monomer-dimer equilibrium (K(D)dimer = 8.0 muM, inta ct enzyme; K(D)dimer = 0.1 muM, large fragment) and that the small fra gment, which displays DNA binding specificity, is a monomer. Dimerizat ion is further supported by line width comparisons in one-dimensional NMR spectra and determinations of the correlation time of the protein. The one-dimensional proton NMR spectra and two-dimensional nuclear Ov erhauser enhancement spectroscopy spectra indicate that the overall st ructure of the two isolated fragments is highly similar to the structu re present in the domains of the intact enzyme. The rotational correla tion time of resolvase, determined from relaxation data obtained from each domain, indicates that the small domain has a limited degree of a dditional motion with respect to the large domain of the enzyme. The m onomer-dimer equilibrium and small domain mobility may assist in the b inding of resolvase to palindromic-type sites with variable spacers an d in subunit exchange during catalysis.