Book of Abstracts: Albany 2003
June 17-21 2003
Conformational Changes in 8-oxoguanine DNA Glycosylase Catalysis
Base excision DNA repair (BER) is the principal pathway responsible for the removal of damaged bases. Key components of the BER pathway are DNA glycosylases, which recognize damaged bases and catalyze their expulsion. One major class of such enzymes, glycosylases/lyases, also catalyzes scission of the DNA backbone following base expulsion. Understanding how BER enzymes recognize and process their substrate has been the subject of extensive research, including X-Ray analyses of such enzymes from which chemical mechanisms of enzyme action have been proposed. For catalysis to occur the damaged nucleotide must be everted from DNA to expose its C1? site to the catalytic machinery of the enzyme. The glycosylase and lyase functions of these enzymes are mechanistically unified through a common, amine-bearing residue on the enzyme.
However, structural analysis alone of protein-nucleic acid interactions at a molecular level cannot provide quantitative estimates of the relative importance of molecular contacts, nor of the relative contributions of strong and weak, or specific and nonspecific, contacts to the total affinity of an enzyme for DNA. Recently our analysis of the interactions involved for a DNA repair enzyme, 8-oxoguanine DNA glycosylase from E. coli (Fpg-protein), with DNA substrates (1) showed that the enzyme specificity is mostly kinetic than thermodynamic in its nature.
We have used presteady-state stopped-flow kinetic methods and protein fluorescence changes to resolve a dynamic questions of repair process catalyzed with 8-oxoguanine DNA glycosylases, E. coli Fpg-protein and human hOgg1 (2). Using the twelve-nucleotide duplex d(CTCTCXCCTTCC) · d(GGAAGGCGAGAG), where where X = G, 8-oxoG, ribose (AP-nucleotide) and tetrahydrofuran (non-cleavable AP-nucleotide), and single-turnover conditions guiding the choice of enzyme concentrations, multiple transient changes in fluorescence were observed, indicating conformational transitions in the protein molecule. The fast phases, where the most pronounced changes in fluorescence occur, were detected as the initial parts of the fluorescence traces. These phases reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformations. The final slower stages corresponding to actual chemistry steps can be seen for cleavable substrates. The fluorescence intensities are restored to the initial values in this case, indicating the decomposition of enzyme-product complex. The intrinsic rate constants of each of the steps were determined using a global fitting procedure.
The research is supported by grants from the Wellcome Trust (UK), the Russian Foundation for Basic Research.
Novosibirsk Institute of Bioorganic Chemistry