Mutational studies of Pa-AGOG DNA glycosylase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum |
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| Authors: | Gondichatnahalli M Lingaraju Andrea E Prota Fritz K Winkler |
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| Institution: | 1. Department of Mathematics and Computer Science, Manchester University, N. Manchester, IN 46962, USA;2. Department of Mathematics Education, Kwandong University, Kanglung, Kanwondo 210-701, South Korea;1. Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas;2. Department of Physics and Astronomy, Rice University, Houston, Texas;3. Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas;4. Department of Medical Physics, The Ottawa Hospital Cancer Centre, Ottawa, Ontario, Canada;5. Crystal Growth Division, Landauer, Inc, Stillwater, Oklahoma;1. Department of Chemistry, Brown University, Providence, RI 02912, United States;2. Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, United States;1. Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan;2. Groupe «Réparation de l''ADN», Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France;3. Novosibirsk State University, Novosibirsk 630090, Russia;4. SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia;1. CRMD, site de Chartres, EA 4229 Université d''Orléans, 21 rue de Loigny la Bataille, 28000 Chartres, France;2. LPiC, EA 2160, Faculté de Pharmacie, Université de Nantes, 9 rue Bias, BP 53508, 44035 Nantes Cedex 1, France;3. IIciMed, EA 1155, Faculté de Pharmacie, Université de Nantes, 9 rue Bias, BP 53508, 44035 Nantes Cedex 1, France;4. Institute for Superconducting and Electronic Materials, University of Wollongong, NSW 2522, Australia |
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| Abstract: | In all organisms studied to date, 8-oxoguanine (GO), an important oxidation product of guanine, is removed by highly conserved GO DNA glycosylases. The hyperthermophilic crenarchaeon Pyrobaculum aerophilum encodes a GO DNA glycosylase, Pa-AGOG (Archaeal GO DNA glycosylase) which has become the founding member of a new family within the HhH-GPD superfamily of DNA glycosylases based on unique structural and functional characteristics. In this study, we made quantitative measurements of the DNA glycosylase activity of Pa-AGOG wild type and some engineered variants under single turnover conditions. The mutagenesis study includes residues Trp222 (W222A and W222F), Trp69 (W69F), Gln31 (Q31S) and Lys147 (K147Q) all of which are involved in GO recognition and Asp172 (D172N and D172Q) and Lys140 (K140Q) that are involved in catalysis. Pa-AGOG prefers GO/G mispairs for both base excision and base excision/β-lyase activities. The mutagenesis studies show that base-stacking between GO and Trp222 is very important for recognition. The contact between Trp69 and the 8-oxo group was found to be dispensable, while that to N7 by Gln31 is indispensable for GO recognition. In contrast to human OGG1 the catalytic mutant, D172Q did not show detectable glycosylase activity. Pa-AGOG mutants K140Q, D172N and D172Q did bind GO containing single-stranded DNA more tightly than double-stranded DNA containing a GO/C base pair. Our studies confirm and extend the unique characteristics of Pa-AGOG, which distinguish it from other mesophilic and thermostable GO DNA glycosylases. |
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