MUTATIONS IN THE ADA O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE CONFERRINGSENSITIVITY TO INACTIVATION BY O-6-BENZYLGUANINE AND 2,4-DIAMINO-6-BENZYLOXY-5-NITROSOPYRIMIDINE

Although the human O-6-alkylguanine-DNA alkyltransferase (AGT) is very sensitive to inactivation by O-6-benzylguanine (BG) or 2,4-diamino-6- benzyloxy-5-nitrosopyrimidine (5-nitroso-BP), the equivalent protein f ormed by the carboxyl terminal domain of the product of the Escherichi a coli ada gene (Ada-C) is unaffected by these inhibitors, This differ ence is remarkable in view of the substantial similarity between these proteins (33% of the residues in the common sequence are identical) a nd is potentially very important since these inhibitors are under deve lopment as drugs to enhance the anti-tumor activity of alkylating agen ts, In order to understand the reason for the resistance of the Ada-C protein, we have made chimeras between Ada-C and AGT sequences and mut ations in the Ada-C protein, expressed the altered proteins in an E. c oli strain lacking endogenous alkyltransferase activity and tested the inactivation of the resulting proteins by BG or 5-nitroso-BP, Chimeri c alkyltransferase proteins were made in which the residues on the ami no side of the cysteine acceptor site came from Ada-C and the residues on the carboxyl side came from AGT and vice versa but these did not s how sensitivity to BG suggesting that resistance is produced by residu es in both segments of the protein, Analysis of the Ada-C mutant prote ins revealed two sites for mutations that confer sensitivity to these inhibitors, One of these was tryptophan-336 and the other was residues lysine-314 and alanine-316. Thus, when the combined mutations of A316 P/W336A were made in the Ada-C sequence, the protein was sensitive to inactivation by BG, This A316P/W336A mutant protein was even more sens itive to S-nitroso-BP and the mutant proteins W336A, K314P/A316P and A 316P could also be inhibited by this drug (in decreasing order of sens itivity) although the control Ada-C and a mutant R335S were not inhibi ted, These results provide strong support for the hypothesis that the resistance of the Ada-C alkyltransferase is due to a steric effect lim iting access to the active site, Insertion of proline residues at posi tions 314 and 316 and removal of the bulky tryptophan residue at posit ion 336 increases the space available at the active site and permits t hese inhibitors to be effective.