Purification and characterization of human 3-methyladenine-DNA glycosylase.
A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli. In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed. All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa. Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E. coli DNA repair proteins. All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA. The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable. In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein.
O'Connor
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Groupe 'Réparation des Lésions Radio- et Chimio-Induites, CNRS URA147/INSERM U140, Institut Gustave-Roussy, Villejuif, France.
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003429:Cross Reactions; D004247:DNA; D045647:DNA Glycosylases; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction
C044502:3-methylguanine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II