Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.
The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-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 (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) 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(50) 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 Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy.
Xu-Welliver
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M
Department 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, USA.
Kanugula
S
S
Loktionova
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NA
Crone
T M
TM
Pegg
A E
AE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D002330:Carmustine / Q000494:pharmacology / Q000633:toxicity; D017124:Conserved Sequence / Q000235:genetics*; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology / Q000633:toxicity; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment; D014162:Transfection
C110997:8-oxo-O(6)-benzylguanine; D000970:Antineoplastic Agents; C064976:O(6)-benzylguanine; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase; D008239:Lysine; D002330:Carmustine