Thermostability and excision activity of polymorphic forms of hOGG1

Abstract

Abstract Objectives Reactive oxygen species (ROS) oxidize guanine residues in DNA to form 7,8-dihydro-oxo-2′-deoxyguanosine (8oxoG) lesions in the genome. Human 8-oxoguanine glycosylase-1 (hOGG1) recognizes and excises this highly mutagenic species when it is base-paired opposite a cytosine. We sought to characterize biochemically several hOGG1 variants that have been found in cancer tissues and cell lines, reasoning that if these variants have reduced repair capabilities, they could lead to an increased chance of mutagenesis and carcinogenesis. Results We have over-expressed and purified the R46Q, A85S, R154H, and S232T hOGG1 variants and have investigated their repair efficiency and thermostability. The hOGG1 variants showed only minor perturbations in the kinetics of 8oxoG excision relative to wild-type hOGG1. Thermal denaturation monitored by circular dichroism revealed that R46Q hOGG1 had a significantly lower Tm (36.6 °C) compared to the other hOGG1 variants (40.9 °C to 43.2 °C). Prolonged pre-incubation at 37 °C prior to the glycosylase assay dramatically reduces the excision activity of R46Q hOGG1, has a modest effect on wild-type hOGG1, and a negligible effect on A85S, R154H, and S232T hOGG1. The observed thermolability of hOGG1 variants was mostly alleviated by co-incubation with stoichiometric amounts of competitor DNA.

Keywords

• DNA repair
• 8-Oxoguanine
• DNA glycosylase
• Mutation
• Cancer