Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases

Abstract

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.

Figure 1

Structures of OG(anti):C(anti) pair and OG(syn):A(anti) mispair in DNA (A). Structures of OG deoxynucleoside and of OG analogs 1–4 (B). Nucleosides are drawn with their preferred glycosidic orientation (i.e., OG,1,2 are syn; 3,4 are anti). The standard numbering scheme for purines and indoles is shown for key positions analyzed in this work.

Figure 2

(A) Representative plot of extent of Fpg catalyzed removal of OG and analogs 1–4 as a function of time under conditions of single-turnover from 30 base pair duplexes containing OG:C (), 1:C(), 2:C (), 3:C (), and 4:C (). Inset shows region of Fpg experiments expanded to 1 minute time scale. Experiments were performed using 200 nM Fpg and 20 nM duplex DNA at 37°C. (B) Representative plot for extent of hOGG1 catalyzed removal of OG and nonpolar OG analogs 1–4 as a function of time under conditions of single-turnover from 30 base pair duplexes containing OG:C (), 1:C(), 2:C (), 3:C (), and 4:C (). Inset shows region of OG: C () fit expanded to 1 minute time scale. Experiments were performed using 200 nM hOGG1 and 20 nM duplex DNA at 37°C. (C) Measurement of Fpg affinity for a DNA duplex containing compound 1:C()pair. The dissociation constant Kd was determined from k_obs measurements as a function of [Fpg] performed under STO conditions with 5 pM duplex DNA.

Figure 3

Histogram illustrating the extent of depurination of G, OG, and analogs 1–4 at position 16 of a 30 base pair duplex normalized to extent of cleavage of G at position 12. Quantitation of data from several experiments provided the relative % extents of cleavage: G, 1.1 ± 0.1; OG, 2.3 ± 0.6; 1, 0.5 ± 0.1; 2, 1.4 ± 0.2; 3, 0.9 ± 0.3; 4, 0.6 ± 0.1.

Figure 4

Hydrogen bonding interactions of OG observed in X-ray crystal structure of K249Q hOGG1 and E3Q Fpg bound to an OG:C containing duplex. DNA is shown in yellow with oxygen (red), nitrogen (blue), and phosphorus (purple). Important residues are labeled and shown in grey with oxygen (red), nitrogen (blue), and sulfur (orange). Hydrogen bonds are shown as dotted lines. (A) The view of the base-specific pocket of hOGG1, showing residues involved in hydrogen bonding interactions in recognition of OG. (B) The view of the base-specific pocket of Fpg, showing residues involved in hydrogen bonding interactions in recognition of OG. Images generated from the pdb file, 1EBM (hOGG1) and 1R2Y (Fpg) from the Worldwide Protein Data Bank based on data from references^,