The study of the site-specific DNA recombination reaction catalyzed by gamma-delta resolvase is of general scientific and medical interest. Complete characterization of this DNA recombinase, the only one to be structurally studied in part to date, will provide a basis for the long term understanding of DNA recombinases in a variety of infectious and antibiotic resistant bacteria, in the human immune system, and in retroviral systems. The reaction catalyzed by gamma-delta resolvase has mechanistic similarities to a topoisomerase reaction but is DNA sequence specific. The resolution of a cointegrate plasmid by resolvase involves four cleavage reactions of the phosphodiester backbone, with conservation of the energy of the phosphodiester bond. The resulting eight ends of DNA are then rejoined through an interaction of two resolvasomes (three dimers of resolvase bound to a res site) to yield two recombinant DNA molecules. The goals of this research are to elucidate mechanistic and structural aspects of the resolvase catalyzed reaction using primarily NMR spectroscopy and other biochemical methods. Structure-function analysis will utilize the domain structure and will be guided by, as well as employ the study of mutants that display specific dysfunctional repressor, recombination, or dimerization activities. The 3D solution structure of the DNA binding domain will be refined by further experiments, and its complex formed through interaction with both the major and minor groove of res site I DNA will be studied. The DNA binding domain containing a linker segment (116-183 fragment) will be structurally characterized. T7 overexpression systems will be developed that allow cost effective 15N/13C labeling of gamma-delta resolvase and its subdomains. Double and triple resonance 3D and 4D NMR will be used to assign intraresidue 1H, 15N, and 13C resonances in the large fragment, and methods that transfer magnetization through the heteronuclear coupling of the peptide bond will be used to sequentially assign the amino acid spin systems. 3D NOESY-HMQC and 4D HMQC-NOESY-HMQC editing with both the 15N and 13C nucleus will be used for obtaining interproton distance information. This research will provide completion of the first structure of a site-specific DNA recombinase, understanding of subdomain protein-protein interactions, and an understanding of subdomain protein-DNA interactions in resolvase. NMR will be used to determine the nature of backbone dynamics, which based on the partial x-ray structural data is required for the function of this enzyme.
| Liu, T; DeRose, E F; Mullen, G P (1994) Determination of the structure of the DNA binding domain of gamma delta resolvase in solution. Protein Sci 3:1286-95 |
