Surprising Repair Activities of Nonpolar Analogs of 8-oxoG Expose Features of Recognition and Catalysis by Base Excision Repair Glycosylases

• Published in: Journal of the American Chemical Society 2012-01, Vol.134 (3), p.1653-1661
• Main Authors: McKibbin, Paige L, Kobori, Akio, Taniguchi, Yosuke, Kool, Eric T, David, Sheila S
• Format: Article
• Language: English
• Subjects: DNA - chemistry; DNA - metabolism; DNA Glycosylases - metabolism; DNA Repair; DNA-Formamidopyrimidine Glycosylase - metabolism; Geobacillus stearothermophilus - chemistry; Geobacillus stearothermophilus - enzymology; Guanine - analogs & derivatives; Guanine - chemistry; Guanine - metabolism; Humans; Models, Molecular
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja208510m

Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.