The excision of 8-oxoguanine (oxoG) by the human 8-oxoguanine DNA glycosylase 1 (hOGG1) base-excision repair enzyme was studied by using the QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculation method and nuclear magnetic resonance (NMR) spectroscopy. The calculated glycosylase reaction included excision of the oxoG base, formation of Lys249-ribose enzyme-substrate covalent adduct and formation of a Schiff base. The formation of a Schiff base with ΔG# = 17.7 kcal/mol was the rate-limiting step of the reaction. The excision of the oxoG base with ΔG# = 16.1 kcal/mol proceeded via substitution of the C1΄-N9 N-glycosidic bond with an H-N9 bond where the negative charge on the oxoG base and the positive charge on the ribose were compensated in a concerted manner by NH3+(Lys249) and CO2-(Asp268), respectively. The effect of Asp268 on the oxoG excision was demonstrated with 1H NMR for WT hOGG1 and the hOGG1(D268N) mutant: the excision of oxoG was notably suppressed when Asp268 was mutated to Asn. The loss of the base-excision function was rationalized with QM/MM calculations and Asp268 was confirmed as the electrostatic stabilizer of ribose oxocarbenium through the initial base-excision step of DNA repair. The NMR experiments and QM/MM calculations consistently illustrated the base-excision reaction operated by hOGG1.

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic v v i Zámek 136 373 33 Nové Hrady Czech Republic

Department of Electrotechnology Electrical Engineering Czech Technical University Technická 2 166 27 Praha Czech Republic

Division of Genome Biology National Cancer Center Research Institute 1 1 Tsukiji 5 chome Chuo ku Tokyo 104 0045 Japan

Faculty of Pharmaceutical Sciences Tokushima Bunri University Nishihama Boji 180 Yamashiro cho Tokushima 770 8514 Japan

Graduate School of Engineering Yokohama National University Tokiwadai 79 5 Hodogaya ku Yokohama 240 8501 Japan

Graduate School of Pharmaceutical Sciences Tohoku University Aobayama Sendai 980 8578 Japan

The Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo námestí 2 166 10 Praha Czech Republic

The Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo náměstí 2 166 10 Praha Czech Republic

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