Conserved residue lysine(165) is essential for the ability of O-6-alkylguanine-DNA alkyltransferase to react with O-6-benzylguanine

The role of lysine(165) in the activity of the DNA repair protein, O-6-alky lguanine-DNA alkyltransferase (AGT), and the ability of ACT to react with t he pseudosubstrate inhibitor, O-6-benzylguanine (BG), was investigated by c hanging this lysine to all other 19 possibilities. All of these mutants (ex cept for K165T, which could not be tested as it was too poorly active for a ssay in crude cell extracts) gave BG-resistant AGTs with increases in the a mount of inhibitor needed to produce a 50 % loss of activity in a 30 min in cubation (ED50) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is com pletely conserved residue in AGTs from many species, and all of the mutatio ns at this site also reduced the ability to repair methylated DNA. The leas t deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED50 value for inactivati on by BG > 200-fold greater than wild-type. Detailed studies of purified K1 65A AGT showed that the rate constant for repair and the binding to methyla ted DNA substrates were reduced by 10-20-fold. Despite this, the K165A muta nt AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at posit ion 165 is needed for optimal activity of AGT towards methylated DNA substr ates and is essential for efficient reaction with BG; and second, even if t he AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkyla ting agents. These results raise the possibility that the conservation of L ys(165) is due to the need for AGT activity towards substrates containing m ore bulky adducts than O-6-methylguanine, They also suggest that alteration s at Lys(165) may occur during chemotherapy with BG and alkylating agents a nd could limit the effectiveness of this therapy.