Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine-DNA alkyltransferase insensitive to O6-benzylguanine

Meng Xu-Welliver, Anthony E. Pegg

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O6-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 Lys165, Leu168 and Leu169. The two positions at which changes led to the largest increase in resistance to BG were Gly156 and Lys165. Eleven mutants at Gly156 Were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys165 found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser159 position (S159I and S159V) were > 80-fold more resistant than wild-type AGT. Eleven active mutants at Leu169 were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys150, 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 Asn157 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.

Original languageEnglish (US)
Pages (from-to)519-526
Number of pages8
JournalBiochemical Journal
Volume347
Issue number2
DOIs
StatePublished - Apr 15 2000

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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