Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O-6-alkylguanine-DNA alkyltransferase insensitive to O-6-benzylguanine

The DNA repair protein, O-6-alkylguanine-DNA alkyltransferase (AGT), is ina ctivated by reaction with the pseudosubstrate, O-6-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents , and BG is aimed at enhancing cancer treatment in clinical trials. Point m utations in a 24 amino acid sequence likely to form the BG-binding pocket w ere identified using a screening method designed to identify BG-resistant m utants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserve d residues Lys(165), Leu(168) and Leu(169). The two positions at which chan ges led to the largest increase in resistance to BG were Gly(156) and Lys(1 65). Eleven mutants at Gly(156) were identified, with increases in resistan ce ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys(1 65) found in the screen (K165S and K165A) showed 620-fold and 100-fold incr eases in resistance to BG. Two mutants at the Ser(159) position (S159I and S159V) were > 80-fold more resistant than wild-type AGT. Eleven active muta nts at Leu(169) were also resistant to BG, but with lower increases (5-86-f old). Fourteen BG-resistant mutants were found for position Cys(150), with 3-26-fold increases in the amount of inhibitor needed to produce a 50 % los s of activity in a 30 min incubation. Six BG-resistant mutants at Asn(157) were found with increases of 4-13-fold. These results show that many change s can render human AGT resistant to BG without preventing the ability to pr otect tumour cells from therapeutic alkylating agents.