Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases.
O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.
Goodtzova
K
K
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033-0850, USA.
Kanugula
S
S
Edara
S
S
Pauly
G T
GT
Moschel
R C
RC
Pegg
A E
AE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004260:DNA Repair; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
C086794:Ada-C protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; C064976:O(6)-benzylguanine; D012965:Sodium Chloride; D006147:Guanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; C067511:Ogt protein, E coli