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外文期刊>The biochemical journal
>Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine–DNA alkyltransferase insensitive to O6-benzylguanine
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Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine–DNA alkyltransferase insensitive to O6-benzylguanine
pThe DNA repair protein, iO/isup6/sup-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, iO/isup6/sup-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lyssup165/sup, Leusup168/sup and Leusup169/sup. The two positions at which changes led to the largest increase in resistance to BG were Glysup156/sup and Lyssup165/sup. Eleven mutants at Glysup156/sup were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lyssup165/sup found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Sersup159/sup position (S159I and S159V) were & 80-fold more resistant than wild-type AGT. Eleven active mutants at Leusup169/sup were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cyssup150/sup, with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asnsup157/sup were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents./p
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