The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)-DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4' of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key DNA repair enzyme that excises 8-oxoguanine, a mutagenic base lesion, from double-stranded DNA. In this study, we crystallographically visualized an intermediate state of the enzymatic reaction. To achieve this, we employed a specifically designed pH-sensitive mutant of hOGG1 and applied a freeze-trapping technique to capture the reaction intermediate. The resulting crystal structure revealed a previously unknown reaction pathway involving a hemiaminal-type intermediate, captured here for the first time. These findings provide new insights into the catalytic mechanism of hOGG1.

Graduate School of Science and Engineering Ibaraki University Hitachi Ibaraki 316 8511 Japan

Institute of Advanced Medical Sciences Tokushima University Kuramoto cho 3 18 15 Tokushima 770 8503 Japan

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo náměstí 2 Praha 160 00 Czech Republic

Laboratory of Analytical Chemistry Faculty of Pharmaceutical Sciences Tokushima Bunri University Yamashiro cho Tokushima 770 8514 Japan

Research and Education Center for Atomic Sciences Ibaraki University Naka Tokai Ibaraki 319 1106 Japan

Zobrazit více v PubMed

Wood  RD, Mitchell  M, Sgouros  J  et al.  Human DNA repair genes. Science. 2001; 291:1284–9. 10.1126/science.1056154. PubMed DOI

Arai  K, Morishita  K, Shinmura  K  et al.  Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage. Oncogene. 1997; 14:2857–61. 10.1038/sj.onc.1201139. PubMed DOI

Kohno  T, Shinmura  K, Tosaka  M  et al.  Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA. Oncogene. 1998; 16:3219–25. 10.1038/sj.onc.1201872. PubMed DOI

Sheng  Z, Oka  S, Tsuchimoto  D  et al.  8-Oxoguanine causes neurodegeneration during MUTYH-mediated DNA base excision repair. J Clin Invest. 2012; 122:4344–61. 10.1172/JCI65053. PubMed DOI PMC

Jacob  KD, Noren  Hooten N, Tadokoro  T  et al.  Alzheimer’s disease-associated polymorphisms in human OGG1 alter catalytic activity and sensitize cells to DNA damage. Free Radic Biol Med. 2013; 63:115–25. 10.1016/j.freeradbiomed.2013.05.010. PubMed DOI PMC

Wang  R, Li  C, Qiao  P  et al.  OGG1-initiated base excision repair exacerbates oxidative stress-induced parthanatos. Cell Death Dis. 2018; 9:628. 10.1038/s41419-018-0680-0. PubMed DOI PMC

Nash  HM, Lu  R, Lane  WS  et al.  The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution. Chem Biol. 1997; 4:693–702. 10.1016/S1074-5521(97)90225-8. PubMed DOI

Nash  HM, Bruner  SD, Scharer  OD  et al.  Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr Biol. 1996; 6:968–80. 10.1016/S0960-9822(02)00641-3. PubMed DOI

Norman  DP, Chung  SJ, Verdine  GL  Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase. Biochemistry. 2003; 42:1564–72. 10.1021/bi026823d. PubMed DOI

Labahn  J, Schärer  OD, Long  A  et al.  Structural basis for the excision repair of alkylation-damaged DNA. Cell. 1996; 86:321–9. 10.1016/S0092-8674(00)80103-8. PubMed DOI

Thayer  MM, Ahern  H, Xing  D  et al.  Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure. EMBO J. 1995; 14:4108–20. 10.1002/j.1460-2075.1995.tb00083.x. PubMed DOI PMC

Piersen  CE, Prince  MA, Augustine  ML  et al.  Purification and cloning of PubMed DOI

Bruner  SD, Norman  DP, Verdine  GL  Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature. 2000; 403:859–66. 10.1038/35002510. PubMed DOI

Bjørås  M, Seeberg  E, Luna  L  et al.  Reciprocal “flipping” underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase. J Mol Biol. 2002; 317:171–7. 10.1006/jmbi.2002.5400. PubMed DOI

Sebera  J, Hattori  Y, Sato  D  et al.  The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine. Nucleic Acids Res. 2017; 45:5231–42. 10.1093/nar/gkx157. PubMed DOI PMC

Fromme  JC, Bruner  SD, Yang  W  et al.  Product-assisted catalysis in base-excision DNA repair. Nat Struct Mol Biol. 2003; 10:204–11. 10.1038/nsb902. PubMed DOI

Matsui  T, Unno  M, Ikeda-Saito  M  Heme oxygenase reveals its strategy for catalyzing three successive oxygenation reactions. Acc Chem Res. 2010; 43:240–7. 10.1021/ar9001685. PubMed DOI

Lai  W, Chen  H, Matsui  T  et al.  Enzymatic ring-opening mechanism of verdoheme by the heme oxygenase: a combined X-ray crystallography and QM/MM study. J Am Chem Soc. 2010; 132:12960–70. 10.1021/ja104674q. PubMed DOI

Unno  M, Ardèvol  A, Rovira  C  et al.  Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from PubMed DOI PMC

Unno  M, Matsui  T, Ikeda-Saito  M  Crystallographic studies of heme oxygenase complexed with an unstable reaction intermediate, verdoheme. J Inorg Biochem. 2012; 113:102–9. 10.1016/j.jinorgbio.2012.04.012. PubMed DOI

Dalhus  B, Forsbring  M, Helle  IH  et al.  Separation-of-function mutants unravel the dual-reaction mode of human 8-oxoguanine DNA glycosylase. Structure. 2011; 19:117–27. 10.1016/j.str.2010.09.023. PubMed DOI

Kabsch  W  Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr. 2010; 66:133–44. 10.1107/S0907444909047374. PubMed DOI PMC

Evans  PR, Murshudov  GN  How good are my data and what is the resolution?. Acta Crystallogr D Biol Crystallogr. 2013; 69:1204–14. 10.1107/S0907444913000061. PubMed DOI PMC

Vagin  A, Teplyakov  A  MOLREP: an automated program for molecular replacement. J Appl Crystallogr. 1997; 30:1022–5. 10.1107/S0021889897006766. DOI

Potterton  E, Briggs  P, Turkenburg  M  et al.  A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr. 2003; 59:1131–7. 10.1107/S0907444903008126. PubMed DOI

Emsley  P, Cowtan  K  Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004; 60:2126–32. 10.1107/S0907444904019158. PubMed DOI

Murshudov  GN, Skubak  P, Lebedev  AA  et al.  REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011; 67:355–67. 10.1107/S0907444911001314. PubMed DOI PMC

Liebschner  D, Afonine  PV, Baker  ML  et al.  Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol. 2019; 75:861–77. 10.1107/S2059798319011471. PubMed DOI PMC

Sadeghian  K, Ochsenfeld  C  Unraveling the base excision repair mechanism of human DNA glycosylase. J Am Chem Soc. 2015; 137:9824–31. 10.1021/jacs.5b01449. PubMed DOI

Zhu  C, Lu  L, Zhang  J  et al.  Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair. Proc Natl Acad Sci USA. 2016; 113:7792–7. 10.1073/pnas.1604591113. PubMed DOI PMC

Schyman  P, Danielsson  J, Pinak  M  et al.  Theoretical study of the human DNA repair protein HOGG1 activity. J Phys Chem A. 2005; 109:1713–9. 10.1021/jp045686m. PubMed DOI

Calvaresi  M, Bottoni  A, Garavelli  M  Computational clues for a new mechanism in the glycosylase activity of the human DNA repair protein hOGG1. A generalized paradigm for purine-repairing systems?. J Phys Chem B. 2007; 111:6557–70. 10.1021/jp071581i. PubMed DOI

Shim  EJ, Przybylski  JL, Wetmore  SD  Effects of nucleophile, oxidative damage, and nucleobase orientation on the glycosidic bond cleavage in deoxyguanosine. J Phys Chem B. 2010; 114:2319–26. 10.1021/jp9113656. PubMed DOI

Šebera  J, Trantírek  L, Tanaka  Y  et al.  Pyramidalization of the glycosidic nitrogen provides the way for efficient cleavage of the N-glycosidic bond of 8-OxoG with the hOGG1 DNA repair protein. J Phys Chem B. 2012; 116:12535–44. 10.1021/jp309098d. PubMed DOI

Kellie  JL, Wilson  KA, Wetmore  SD  Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase. Biochemistry. 2013; 52:8753–65. 10.1021/bi401310w. PubMed DOI

Šebera  J, Trantírek  L, Tanak  Y  et al.  The activation of DOI

Dracinsky  M, Šála  M, Klepetářová  B  et al.  Benchmark theoretical and experimental study on PubMed DOI

Liu  L, Cottrell  JW, Scott  LG  et al.  Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme. Nat Chem Biol. 2009; 5:351–7. 10.1038/nchembio.156. PubMed DOI PMC

Tanaka  Y, Yamanaka  D, Morioka  S  et al.  Physicochemical characterization of the catalytic unit of hammerhead ribozyme and its relationship with the catalytic activity. Biophysica. 2022; 2:221–39. 10.3390/biophysica2030022. DOI

Tomar  R, Minko  IG, Sharma  P  et al.  Base excision repair of the PubMed DOI PMC

Schanda  P, Kupce  E, Brutscher  B  SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR. 2005; 33:199–211. 10.1007/s10858-005-4425-x. PubMed DOI

Vollhardt  KPC, Schore  NE  Organic Chemistry : Structure and Function. 2018; 8th ednNew York: W.H. Freeman & Co. Ltd.

Bruice  PY  Organic Chemistry. 2007; 5th ednLondon: Pearson Education, Inc.