TY - JOUR
T1 - Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine
AU - Xu-Welliver, Meng
AU - Kanugula, Sreenivas
AU - Loktionova, Natalia A.
AU - Crone, Tina M.
AU - Pegg, Anthony E.
N1 - Copyright:
Copyright 2004 Elsevier Science B.V., Amsterdam. All rights reserved.
PY - 2000/4/15
Y1 - 2000/4/15
N2 - The role of lysine165 in the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O6-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED50) from 100-fold (K 165A) to 2400-fold (K165F). Lys165 is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least 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 ED,, value for inactivation by BG > 200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys165 is due to the need for AGT activity towards substrates containing more bulky adducts than O6-methylguanine. They also suggest that alterations at Lys165 may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy.
AB - The role of lysine165 in the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O6-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED50) from 100-fold (K 165A) to 2400-fold (K165F). Lys165 is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least 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 ED,, value for inactivation by BG > 200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys165 is due to the need for AGT activity towards substrates containing more bulky adducts than O6-methylguanine. They also suggest that alterations at Lys165 may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy.
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U2 - 10.1042/0264-6021:3470527
DO - 10.1042/0264-6021:3470527
M3 - Article
C2 - 10749683
AN - SCOPUS:0034654658
VL - 347
SP - 527
EP - 534
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 2
ER -