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. 2006 Mar 20;34(5):1620-32.
doi: 10.1093/nar/gkl060. Print 2006.

Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

Jeff W Hill  1 , Michele K Evans
Affiliations

Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

Jeff W Hill et al. Nucleic Acids Res. .

Abstract

Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.

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Figures

Figure 1
Figure 1
SDS–PAGE analysis of purified proteins. Lane 1, 0.5 µg wild-type OGG1; lane 2, 0.5 µg S326C OGG1; lane 3, 0.5 µg APE1, lane M, molecular weight marker. Proteins were purified as described in Materials and Methods and electrophoresed on a 4–20% acrylamide gel.
Figure 2
Figure 2
Kinetics of wild-type and S326C OGG1. Wild-type and S326C OGG1 (2.5 nM) were incubated with increasing amounts (3.25–100 nM) of duplex 8-oxoguanine substrates having C (A), T (B), G (C) or A (D) opposite 8-oxoG, or with a substrate having an abasic site opposite C (E). Glycosylase reactions with 8-oxoguanine paired with C, T and G, were incubated for 15 min at 37°C. AP-lyase reactions with the AP·C substrate and glycosylase reactions with 8-oxoG opposite A were incubated for 1 h at 37°C. Reactions were terminated and analyzed as described in Materials and Methods. Data points are means of three independent experiments with standard deviation.
Figure 3
Figure 3
EMSAs of wild-type and S326C OGG1. DNA damage binding affinities of wild-type and S326C OGG1 were measured by incubation with DNA substrates containing 8-oxoG·C (A), 8-oxoG·T (B), 8-oxoG·G (C), 8-oxoG·A (D) and AP·C (E). (F) Identical to the experiment in (A), with the addition of 1 mM DTT. Gel shifts of glycosylases were performed as described in Materials and Methods. In all panels, lane 1, no enzyme; lane 2, wild-type OGG1; lane 3, S326C OGG1.
Figure 4
Figure 4
Dimerization of polymorphic S326C OGG1. (A) Wild-type (lanes 1 and 2) and S326C OGG1 (lanes 3 and 4) at a concentration of 5 µM were incubated with 1 mM of BM[PEO]4 cross-linker in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 10 µM duplex 8-oxoG·C substrate. Lane M, molecular weight marker. Reactions were analyzed by SDS–PAGE on 4–20% acrylamide gels. (B) Wild-type OGG1 (2.5 µM) in lanes 1–4 was incubated with 1 mM BM[PEO]4 in the presence of 0 (lane 1), 1.25 µM (lane 2), 2.5 µM (lane 3) and 5 µM (lane 4) S326C OGG1.
Figure 5
Figure 5
Size-exclusion chromatographic analysis of wild-type and S326C OGG1. (A) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min (B). Identical runs were performed with 100 µg polymorphic S326C OGG1 (C) or 100 µg of both wild-type and S326C OGG1 together (D).
Figure 6
Figure 6
Effect of AP-endonuclease on wild-type and S326C OGG1. (A) Glycosylase activities of wild-type (black columns) and S326C OGG1 (grey columns) (12.5 nM) were measured with an 8-oxoG·C substrate (250 nM) with or without an equimolar amount of APE1, in the presence or absence of 1 mM DTT. Inset, actual data. Lanes 1–4, wild-type OGG1; lanes 5–8, S326C; lanes 2, 4, 6 and 8, plus 1 mM DTT; lanes 3–4 and 7–8 plus 12.5 nM APE1. Inset, actual data. (B) Binding of OGG1 enzymes to an abasic site substrate after adding APE1 was measured by EMSA. Wild-type and S326C OGG1 (5 nM) were incubated with an AP site-containing duplex substrate (5 nM) in the presence of 1 mM MgCl2 at 37°C for 1 min prior to adding an equimolar amount of APE1 and incubating for an additional 1 min at 37°C. Lane 1, no enzyme; lanes 2 and 3, wild-type OGG1; lanes 4 and 5, S326C OGG1; lanes 3 and 5, plus APE1. Inset, actual data. (C) Borohydride trapping of OGG1 enzymes. Lane 1, molecular weight marker. Trapping of wild-type (lanes 2 and 3) and S326C OGG1 (lanes 4 and 5) with an 8-oxoG·C substrate in the presence (lanes 3 and 5) and absence (lanes 2 and 4) of AP-endonuclease as described in Materials and Methods. Inset, actual data.
Figure 7
Figure 7
Binding and catalysis by OGG1 deletion mutants. (A) Amino acid sequence of the C-termini of wild-type, wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1. (B) Wild-type OGG1 (lane 1) was reacted with an 8-oxoG·C substrate as described in Figure 6. Identical reactions were carried out for wild-type CΔ19 (lane 2), S326C (lane 3), S326C CΔ19 (lane 4) and CΔ20 OGG1 (lane 5). (C) Graphical representation of actual data shown in (B). (D) Binding of OGG1 enzymes to an 8-oxoG·C substate was measured by electrophoretic shift assay as described in Figure 3. Lane 1, no enzyme; lane 2, wild-type; lane 3 wild-type CΔ19; lane 4, S326C; lane 5, S326C CΔ19; and lane 6 CΔ20 OGG1. (E) Graphical representation of actual data shown in (D). Inset, dissociation constants calculated from data shown in (D).
Figure 8
Figure 8
Characterization of wild-type and S326C OGG1 expressed in human cells. (A) Comparision of 8-oxoguanine glycosylase activites of HeLa nuclear extracts from cells transfected with pCMV vector (lane 1), or pCMV vector expressing N-terminally FLAG-tagged wild-type (lane 2) or S326C OGG1 (lane 3). Left inset; Anti-FLAG western blot of 2 µg of nuclear extract prepared from HeLa cells transfected with pCMV vector (lane 1), pCMV wt (lane 2) or PCMV S326C (lane 3). Right inset; actual data shown graphically in (A). (B) Anti-FLAG western blot of 4 µg of nuclear extract from cells transfected with pCMV vector (lanes 1 and 2), pCMV wt (lanes 3 and 4) or pCMV S326C (lanes 5 and 6). Nuclear extracts were incubated at 0°C overnight with (lanes 2, 4 and 6) and without (lanes 1, 3 and 5) 2 mM BM[PEO]4.
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