Factors influencing the repair of the mutagenic lesion O6-methylguanine in DNA by human O6-methylguanine-DNA methyltransferase.
Oligodeoxynucleotides of various chain lengths (p(Bp)nB, n < or = 9) and the eight possible dinucleotide phosphates (pm6GpB and pBpm6G), each containing a single O6-methylguanine residue (m6G), were used to study the repair kinetics of this lesion by the cloned DNA repair proteins; human 21 kDa O6-methylguanine-DNA methyltransferase (MGMT), human 43 kDa glutathione-S-transferase fused MGMT (GSTMGMT) and the Escherichia coli 39 kDa ada protein. The observed second-order repair rate constants are dependent upon both the chain length of the oligonucleotide substrates for all three proteins and in the case assuming O6-methylguanine is similar to B). The differences observed in the ratios of the rate constants for the substrates with five and four base residues; 125 for the E. coli 39 kDa ada protein, 640 for the human MGMT and 27,800 for the human fusion protein GSTMGMT, suggest that the pentanucleotide phosphate containing this lesion is the "optimal" substrate for the proteins. Surprisingly, the human GSTMGMT is shown to be more effective in the repair of longer substrates with the second-order repair rate constants for TATA-Cm6GTATA being 6.16 x 10(6) for GSTMGMT, 2.00 x 10(6) for MGMT and 0.27 x 10(6) M-1 s-1 for the E. coli 39 kDa ada protein. Thus, the presence of an additional protein domain at the N terminus of human MGMT can alter its selectivity towards certain substrates. Although a number of peptide domains are conserved between the E. coli 39 kDa ada protein and phosphates can also be used to explain the observed sequence specific repair of this lesion within certain DNA sequences.
Chemical Carcinogenesis Laboratory, National University of Singapore.
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Liem
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Wong
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Lim
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Li
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D004260:DNA Repair / Q000502:physiology*; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase