How DNA repair proteins distinguish between the rare sites of damage and the vast expanse of normal DNA is poorly understood. Recognizing the mutagenic lesion 8-oxoguanine (oxoG) represents an especially formidable challenge, because this oxidized nucleobase differs by only two atoms from its normal counterpart, guanine (G). Here we report the use of a covalent trapping strategy to capture a human oxoG repair protein, 8-oxoguanine DNA glycosylase I (hOGG1), in the act of interrogating normal DNA. The X-ray structure of the trapped complex features a target G nucleobase extruded from the DNA helix but denied insertion into the lesion recognition pocket of the enzyme. Free energy difference calculations show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site. The structure reveals a remarkably effective gate-keeping strategy for lesion discrimination and suggests a mechanism for oxoG insertion into the hOGG1 active site.
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We are grateful to Y. Korkhin for help in data collection and processing. We thank Enanta Pharmaceuticals for use of their X-ray instrumentation. We acknowledge the entire staff at MACCHESS, especially C. Heaton and B. Miller, and NSLS X4A for assistance in data collection and processing. We thank D. Jeruzalmi and C. Fromme for valuable discussions. This work was supported by grants from the NIH to G.L.V. and M.K.Author contributions A.B. was responsible for performing the structural and biochemical experiments described herein, whereas W.Y. performed the computational simulations.
The authors declare that they have no competing financial interests.
Data collection and model statistics. (DOC 54 kb)
Figure captions for Supplementary Figures S1-S4. (DOC 35 kb)
Additional details on computational methods and data collection and structure solution. (DOC 56 kb)
Structural validation of the trapping strategy and crosslinking biochemistry. (PDF 658 kb)
Electron density map around the G in the exo- site and the crosslinked C in the G-complex. (JPG 550 kb)
Structural representations of the carbonyl of Gly42 and different nucleobases used in this study. (JPG 149 kb)
Electron density maps around the analogs in the crosslinked complexes. (JPG 767 kb)
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Banerjee, A., Yang, W., Karplus, M. et al. Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA. Nature 434, 612–618 (2005). https://doi.org/10.1038/nature03458
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