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.