Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Kinetic and thermodynamic basis for damaged bases excision by 8-oxoguanine-DNA glycosylases
DNA glycosylases play the opening act in a highly conserved process for excision of damaged bases from DNA called the base excision repair pathway (BER). DNA glycosylases attend to a wide variety of lesions arising from both endogenous and exogenous factors. The types of damage include alkylation, oxidation, and hydrolysis. A major DNA oxidation product is 8-oxoguanine (8-oxoG), a base with a high mutagenic potential. In bacteria, this lesion is repaired by formamidopyrimidine-DNA glycosylase (Fpg), while in the case of humans this function belongs to 8-oxoguanine-DNA glycosylase (OGG1). We have attempted a comprehensive characterization of 8-oxoG recognition by DNA glycosylases. First, we have obtained thermodynamic parameters for melting of DNA duplexes containing 8-oxoG in all possible nucleotide contexts. The energy of stacking interactions of 8-oxoG was in strict dependence on 8-oxoG nucleotide environment, which may affect the recognition of damage and the efficiency of eversion of 8-oxoG from DNA helix by glycosylases. Next, we established how the flexibility of DNA context affects damage recognition by these enzymes (Kirpota et al., 2011). Then, we have found that DNA containing 8-oxoG next to a single strand break provides a good substrate for Fpg, as soon as all structural phosphate residues are maintained. Using site-directed mutagenesis, we have addressed the functions of many previously unstudied amino acid residuess that were predicted to be important for Fpg activity by molecular dynamics simulation and phylogenetic analysis. Of note, many substitutions abolished the excision of 8-oxoG but did not affect the cleavage efficiency of abasic substrates. Finally, we investigated the contribution of separated structural domains of Fpg to specific enzyme-substrate interaction. Surprisingly, despite the absence of the catalytic domain, C-terminal domain of Fpg possessed a low residual ability to recognize and cleave abasic substrates. Our study sheds light on mechanism details of Fpg and OGG1 activity, with the ultimate goal of understanding how binding energy can be spent by these enzymes for catalysis.
This work was supported by RAS Presidium (Molecular and Cellular Biology program), RFBR (11-04-00807, 12-04-33231), Russian Ministry of Science and Education.
O. O. Kirpota, A. V. Endutkin, M. P. Ponomarenko, P. M. Ponomarenko, D. O. Zharkov., G. A. Nevinsky (2011). Thermodynamic and kinetic basis for recognition and repair of 8- oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase. Nucleic Acids Res 39, 4836–4850.
SB RAS Institute of Chemical Biology and Fundamental Medicine