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 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.
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Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
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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
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