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.
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Research Article|
April 10 2000
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;
Meng XU-WELLIVER
1Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, 500 University Drive, Hershey, PA 17033-0850, U.S.A.
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Anthony E. PEGG
Anthony E. PEGG
1
1To whom correspondence should be addressed (e-mail aep1@;psu.edu).
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Publisher: Portland Press Ltd
Received:
November 09 1999
Revision Received:
January 18 2000
Accepted:
February 08 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 347 (2): 519–526.
Article history
Received:
November 09 1999
Revision Received:
January 18 2000
Accepted:
February 08 2000
Citation
Meng XU-WELLIVER, Anthony E. PEGG; Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine–DNA alkyltransferase insensitive to O6-benzylguanine. Biochem J 15 April 2000; 347 (2): 519–526. doi: https://doi.org/10.1042/bj3470519
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