Gregory P. Mullen - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

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.

Mammalian DNA polymerase beta catalyzes abasic site excision and nucleotidyl transfer in DNA repair and replication in mammalian cells. The enzyme is central to the repair of environmentally induced DNA damage in humans. Characterization of the structural mechanism and the factors leading to DNA replication fidelity by beta-Pol are highly important for understanding the function and/or dysfunction of this enzyme. Misincorporations by beta-Pol within single-stranded DNA prone to replication errors relate directly to the structural mechanism of template-primer binding or DNA adduct binding by the enzyme and potentially lead to mutations within genomic DNA and disease. The solution structure, DNA interactions, and backbone dynamics of the N- terminal (residues 2-87) and C-terminal (residues 88-334) domains of beta-Pol are being characterized by nuclear magnetic resonance (NMR) methods. The N-terminal and C-terminal domains are readily obtained with 15N/13C labeling in yields of 40-50 mg of protein domain from 2 liters of culture medium. Studies completed for the N-terminal domain have included the determination of a highly refined solution structure, characterization of the template ssDNA interaction interface, and determination of the backbone dynamics. Backbone dynamics of DNA complexes will be studied. The products of abasic site excision by the N-terminal domain will be determined. The Schiff's base lysine, that contributes to abasic site excision activity, and the pKas of catalytic side chains will be determined by NMR. The results of these experiments will be compared to the findings for the K35A, K68A, and K72A mutants. Binding and structural studies will be directed toward the N-terminal domain in complex with a 9-mer duplex DNA containing an abasic site analogue. Multidimensional NMR of the 15N/13C and the 15N/13C/2H labeled C-terminal domain are proposed for determination of the solution secondary structure, backbone dynamics, and conformational flexibility of the polymerase. The proposed studies are aimed at understanding at a solution structural level the multiple activities in DNA polymerase beta function and replication fidelity.

DESCRIPTION: (adapted from the applicant's abstract) Xray cross complementing protein 1 (XRCC1) participates in the repair of DNA damage and, in mice, has been indicated to also be required for embryonic development. XRCC1 interacts with the DNA repair proteins poly-ADP-ribose polymerase (PARP), DNA polymerase (beta-Pol), and DNA ligase III. The complex formed between XRCC1, beta-Pol and DNA ligase III repairs incised abasic sites in the final three steps of the base excision repair pathway. The N-terminal domain of XRCC1 has been shown to bind specifically to single-strand break DNA and its structure has recently been determined by high resolution NMR methods. This proposal seeks to characterize the molecular mechanism of the interaction of the N-terminal domain of XRCC1 with DNA substrate and with beta-Pol. Mutagenesis methods, in combination with biochemical assays, will be used to elucidate those residues that contribute to binding. The existing NMR-based structure and chemical shift perturbation map of the binding surface will guide the mutagenesis. The full range of DNA substrate specificity will also be explored. Backbone dynamics will be characterized and correlated with functional binding sites. The 40 kDa complex, involving the N-terminal domain of XRCC1, DNA substrate and beta-Pol, will be structurally characterized using NMR-based methods.