SUNY at Albany
June 19-23, 2001
Dynamics of Escherichia coli Fpg-protein interaction with DNA substrates
The cellular nucleotide base, 8-oxoguanine (8-oxoG), is the main product of DNA oxidation by reactive oxygen species generated in cells under UV and ionizing irradiation. The formation of 8-oxoG, a strong endogenic mutagen, leads to mutation of a GC to an AT base pair. This damaged nucleotide is repaired by the enzyme 8-oxoG-glycosylase, also called formamidopyrimidine glycosylase (Fpg-protein). This enzyme isolated from E.coli has three types of activities: hydrolysis of the N-glycosidic bond (DNA glycosylase), b-elimination (AP lyase) and d-elimination. As a result of these functions the oxidized guanosine residues are excised from DNA chain.
Previously we have shown that Fpg-protein interacts with specific and nonspecific oligodeoxynucleotide substrates (1). As the relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude, the high specificity of the functioning enzyme may lie in the catalytic rate constant. The interaction of enzyme with non-specific and specific sites on the substrate, as well as the catalytic process itself, must be accompanied by conformational transitions of the enzyme.
To elucidate structural changes during the repair process we have studied the interaction of 8-oxoguanine-containing 12-mer duplex, d(pCTCTCoxoGCCTTCC): d(pGGAAGGCGAGAG), with Fpg-protein. The kinetic pathway of the reaction was determined by following the transient changes in the intrinsic Fpg-protein fluorescence using stopped-flow. The intrinsic rate constants were fitted to a minimal kinetic model of the reaction pathway, containing four conformational transitions. The first phase of the process, interpreted as a bimolecular encounter of the DNA with the protein, was very fast and was accompanied by a large fluorescence change. The subsequent steps were much slower. In accordance with the enzyme reaction mechanism proposed in the literature (2), it was suggested that second phase represents sliding (migration) of the protein along the DNA chain to the specific site(s). The third and fourth phases detected by stopped flow fluorescence represent the catalytic stages of removal of the base and sugar moieties of the DNA lesion, respectively.
Acknowledgments: The research is supported by grants from the Wellcome Trust (UK), the Russian Foundation for Basic Research and the Siberian Division of Russian Academy of Sciences.
O. Fedorova (1)*, V. Koval (1), A. Ishchenko (1), N. Vasilenko (1), G. Nevinsky (1), K. Douglas (2)
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia (1); School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK (2)