title: Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.
abstract: A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.
authors: Department of Biological Chemistry, School of Medicine, University of Maryland at Baltimore, 21201.
authors: J P
authors: JP
authors: McGoldrick
authors: Y C
authors: YC
authors: Yeh
authors: M
authors: M
authors: Solomon
authors: J M
authors: JM
authors: Essigmann
authors: A L
authors: AL
authors: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001482:Base Composition; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D045647:DNA Glycosylases; D004260:DNA Repair; D017886:Durapatite; D004926:Escherichia coli / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013950:Thymus Gland / Q000378:metabolism
chemical_list: D000906:Antibodies; D009838:Oligodeoxyribonucleotides; D017886:Durapatite; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase