Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA

Abstract

Adenine paired with 8-hydroxyguanine (oh8G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His6-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh8G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q324 and 2-Q310 proteins with type 1-H324 and 2-H310 proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh8G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh8G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh8G, was proportionally reduced. The glycosylase activity on A:oh8G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh8G under physiological salt concentrations.