Purification and characterization of human 3-methyladenine-DNA glycosylase
- 11 December 1993
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 21 (24) , 5561-5569
- https://doi.org/10.1093/nar/21.24.5561
Abstract
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 Nterminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The fulllength 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-methyladenine, 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 KCI), phi (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: K m 9 nM and k cat 10 min −1 - 7-methylguanine: K m 29 nM and k cat 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˚C is less than that of the truncated protein.Keywords
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