The overall objective of proposed research is to use presteady- state kinetics and equilibrium titration techniques to elucidate the mechanism of cognate-site (CATT) recognition by the Avian Sarcoma Virus (ASV) integrase (IN) and to define the structure- function relationship between recognition and regulation of catalytic activity. Kinetic time courses using model oligonucleotides show clear presteady-state bursts during the approach to steady-state. A family of model substrates differing in the number of cognate-sites and their configurational arrangement have been designed and synthesized. Analysis of the burst rates, amplitudes, and reaction products shows that IN forms a variety of distinct reactive complexes with DNA. The first specific of the project is to quantify burst rates and amplitudes and their concentration dependencies in order to identify, catalog, and quantitate all such catalytically competent complexes. In parallel, equilibrium titrations will be performed and analyzed using statistical thermodynamic models to predict equilibrium population distributions of different IN-DNA complexes whose concentration dependencies can then be compared with those of the functionally defined catalytically competent complexes. These comparisons quantitatively elucidate relationships between quaternary structure, e.g. DNA binding stoichiometry or IN oligomeric state, and catalytic activity. It is anticipated that acquisition of such information will lead to recursively improved newer generations of oligonucleotides. A second longer term specific aim is to determine a complete minimal kinetic scheme for the physiologically relevant 3'-end processing reaction - i.e. hydrolytic removal of terminal TT from the cognate CATT. Current understanding of specific aim 1 indicates a configurational requirement for simultaneously binding of 2 cognate-sites. Initial efforts will be focused on oligonucleotide design to achieve: 1) functional optimization - maximum burst amplitude and rate and 2) structural optimization - most accurate representation of physiological reaction. Stopped- flow flurimetry will then be used to elucidate DNA binding reactions, protein conformational and/or oligomeric state changes induced by DNA binding, as well as reaction kinetics using fluorescent substrates. Reaction kinetics will also be monitored using rapid chemical quenched-flow techniques to perform single- turnover and pulse-chase experiments. These presteady-state techniques provide a powerful arsenal of tools for delineating reaction intermediates, enzyme conformational changes, and internal equilibria. The third specific is to extend these mechanistic studies to substrates with non-optimally arranged cognate-site configurations that are functionally impaired. Direct comparison of the kinetic schemes for these substrates with that of the optimized substrate directly elucidates the structure-function relationship between cognate-site recognition and regulation of catalysis.
