title: Differential DNA recognition and glycosylase activity of the native human MutY homolog (hMYH) and recombinant hMYH expressed in bacteria.
abstract: Human MutY homolog (hMYH), an adenine DNA glycosylase, can effectively remove misincorporated adenines opposite template G or 8-oxoG bases, thereby preventing G:C-->T:A transversions. Human cell extracts possess the adenine DNA glycosylase activity of hMYH and can form protein-DNA complexes with both A/G and A/8-oxoG mismatches. hMYH in cell extracts was shown to be the primary binding protein for A/G- and A/8-oxoG-containing DNA substrates by UV cross-linking. However, recombinant hMYH expressed in bacteria has much weaker glycosylase and substrate-binding activities towards A/G mismatches than native hMYH. Moreover, the protein-DNA complex of bacterially expressed hMYH migrates much faster than that of native hMYH in a non-denaturing polyacrylamide gel. Dephosphorylation of native hMYH reduces the glycosylase activity on A/G more extensively than on A/8-oxoG mismatches but does not alter the gel mobility of the protein-DNA complex. Our results suggest that hMYH in human cell extracts may be associated with other factors in the protein-DNA complex to account for its slower mobility in the gel. hMYH and apurinic/apyrimidinic endonuclease (hAPE1) co-migrate with the protein-DNA complex formed by the extracts and A/8-oxoG-containing DNA.
authors: Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
authors: Y
authors: Y
authors: Gu
authors: A L
authors: AL
authors: Lu
mesh_terms: D000225:Adenine / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics; D015153:Blotting, Western; D002457:Cell Extracts; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
chemical_list: D002457:Cell Extracts; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D006151:Guanosine; C046215:8-hydroxyguanosine; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D000225:Adenine