The goal of this research is to understand how human 06-methylguanine-methyltransferase (06meG-MT) recognizes and repairs the DNA alkylation product 06-methylguanine (06meG) and the similar minor product 04-methylthymine (04meT). The repair of these modified bases by 06meG-MT helps protect cells from the mutagenic consequences of alkylation damage. The protein acts by transferring a methyl group (or other alkyl group) from DNA to itself in a suicide reaction, using a cysteine as the alkyl-acceptor. Our general approach is: (i) Express human 06meG-MT in E. coli and purify the protein. (ii) Identify mutant proteins that are defective in the recognition of methylated DNA or in the methyltransfer reaction. Mutants will be obtained by site-directed mutagenesis of the conserved, polar residues that are most likely to be functionally important. A procedure for selection of non-functional 06meG-MT mutants will also be investigated. (iii) Biochemically characterize wild-type and mutant proteins. This includes analysis of methyltransfer kinetics, measurement of the pKa for protonation of the active site cysteine, and determination of the O6meG-DNA binding properties of methyltransfer-defective mutants. (iv) Co-crystallize a methyltransfer-defective mutant of the protein with methylated DNA as a preliminary step in the determination of its structure. These studies will help in understanding how O6meG-MT reduces the mutagenic and carcinogenic effects of an important class of mutagen.
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| Paalman, S R; Sung, C; Clarke, N D (1997) Specificity of DNA repair methyltransferases determined by competitive inactivation with oligonucleotide substrates: evidence that Escherichia coli Ada repairs O6-methylguanine and O4-methylthymine with similar efficiency. Biochemistry 36:11118-24 |
| Brown, L R; Deng, J; Noll, D M et al. (1997) Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase: renaturation and rapid purification of the protein. Protein Expr Purif 9:337-45 |
| Paalman, S R; Noll, D M; Clarke, N D (1997) Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine. Nucleic Acids Res 25:1795-801 |
