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. 2014 Apr 25;447(1):12-8.
doi: 10.1016/j.bbrc.2014.03.044. Epub 2014 Mar 27.

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

M P Kaur  1 , E J Guggenheim  1 , C Pulisciano  1 , S Akbar  1 , R M Kershaw  1 , N J Hodges  2
Affiliations

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

M P Kaur et al. Biochem Biophys Res Commun. .

Abstract

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.

Keywords: BiFC visualisation; Dimer; OGG1; Oxidative stress.

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Figures

Fig. 1
Fig. 1
(A) Induction of oxidative stress and (B) depletion of reduced glutathione (GSH) in A549 cells following treatment with BSO (1000 μM, 24 h). The results represent the mean of four independent experiments (±SD, n = 4) carried out in duplicate, ∗∗∗ and ∗∗ significantly different from controls (Student t-test), P < 0.001 and 0,01 respectively.
Fig. 2
Fig. 2
(A) Principle of the BiFC assay: Cultured A549 cells were co-transfected with YC- and YN-YFP hOGG1 constructs. In the event of hOGG1 dimer formation the YC and YN fragments of YFP come into close physical proximity resulting in YFP fluorescence, which can be detected by confocal microscopy. (B) BiFC fluorescence is only observed in cells transfected with Cys326 N/C and only under conditions of oxidative cellular oxidative stress. (A) and (B) control vectors, (C) and (D) Ser326 N and C, (D) and (E) Cys326 N and C. (A), (C) and (E) untreated, (B), (D) and (E) treated with BSO (1000 μM, 24 h). Scale bar is 10 μm. In all images, nuclear DNA is counterstained with Hoechst 33258.
Fig. 3
Fig. 3
(A) Higher magnification representative images of BiFC fluorescence in Cys326 N/C transfected cells following treatment with BSO (1000 μM, 24 h). Scale bar is 10 μm, nuclear DNA is counterstained with Hoechst 33258. (B) Three-dimensional distribution of yellow BiFC fluorescence in the nucleus of a cell transfected with Cys326-OGG1 vectors following treatment with BSO (1000 μM, 24 h). Nuclei are counterstained with Hoechst 33258.
Fig. 4
Fig. 4
(A) Inhibition of BiFC fluorescence in Cys326 OGG1 transfected cells treated with BSO (1000 μM), 24 h after co-incubation with the antioxidant DMSO (1% v/v) or addition of the disulphide reducing agents β-mercaptoethanol (1 mM) of dithiothreitol (1 mM) for the final four hours of incubation. (B) The bar graph represents the average number of BiFC fluorescent foci per cell ± SD for three independent fields of view; the average number of cells per field was 57.3 ± 13.33. ∗∗∗ Significantly different from BSO treated cells alone (P < 0.001, t-test).
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