Differential DNA recognition and glycosylase activity of the native human MutY homolog (hMYH) and recombinant hMYH expressed in bacteria

Human MutY homolog (hMYH), an adenine DNA glycosylase, can effectively remo ve misincorporated adenines opposite template G or 8-oxoG bases, thereby pr eventing G:C-->T:A transversions. Human cell extracts possess the adenine D NA glycosylase activity of hMYH and can form protein-DNA complexes with bot h A/G and A/8-oxoG mismatches, hMYH in cell extracts was shown to be the pr imary binding protein for A/G- and A/8-oxoG-containing DNA substrates by UV cross-linking. However, recombinant hMYH expressed in bacteria has much we aker glycosylase and substrate-binding activities towards A/G mismatches th an native hMYH. Moreover, the protein-DNA complex of bacterially expressed hMYH migrates much faster than that of native hMYH in a non-denaturing poly acrylamide gel. Dephosphorylation of native hMYH reduces the glycosylase ac tivity on A/G more extensively than on A/8-oxoG mismatches but does not alt er the gel mobility of the protein-DNA complex. Our results suggest that hM YH in human cell extracts may be associated with other factors in the prote in-DNA complex to account for its slower mobility in the gel. hMYH and apur inic/apyrimidinic endonuclease (hAPE1) co-migrate with the protein-DNA comp lex formed by the extracts and A/8-oxoG-containing DNA.