REPAIR OF O-6-BENZYLGUANINE BY THE ESCHERICHIA-COLI ADA AND OGT AND THE HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASES

O-6-Methylguanine is removed from DNA via the transfer of the methyl g roup to a cysteine acceptor site present in the DNA repair protein O-6 -alkylguanine-DNA alkyltransferase. The human alkyltransferase is inac tivated by the free base O-6-benzylguanine, raising the possibility th at substantially larger alkyl groups could also be accepted as substra tes, However, the Escherichia coli alkyltransferase, Ada-C, is not ina ctivated by O-6-benzylguanine, The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations conver ting Ala(316) to a proline (A316P) and Trp(336) to alanine (W336A) or glycine (W336G), These changes increase the space at the active site o f the protein where Cys(321) is buried and thus permit access of the O -6-benzylguanine inhibitor, Reaction of the mutant A316P/FY336A-Ada-C with O-6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ad a-C protein activates the protein, The Ada-C protein was able to repai r O-6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alky ltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases react ed 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 n ot prevent binding of the substrate in the correct orientation for alk yl group transfer.