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Differential inactivation of mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine.

ABSTRACT: The action of O6-benzylguanine (O6-BzlG) on recombinant mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases (ATase; EC 2.1.1.63; methylated-DNA-protein-cysteine methyltransferase) was compared by preincubation of these proteins with the base, followed by measurement of residual ATase activity using [3H]methylated substrate DNA. All of the mammalian proteins examined were inactivated by O6-BzlG (Chinese hamster: I40, 0.04 microM; human and rat: I40, 0.06 microM); however, the murine ATase was substantially more resistant requiring 4-5 fold higher concentrations of O6-BzlG to achieve the same levels of inactivation (I40, 0.28 microM). A similar differential inactivation was seen with human and murine ATases when extracts of 3T6 (murine) cells and Raji (human) cells were compared. Of the two E. coli ATase proteins, only the ogt-encoded protein was inactivated, but approximately 400 times more O6-BzlG was required to achieve a level of inactivation similar to that seen with the human protein (I40, 24.8 microM). When O6-BzlG was present in an oligonucleotide, the differential effect on the murine, human and ogt-encoded ATases was not seen and only the ada-encoded ATase remained refractory under the conditions used.

SUBMITTER: Elder RH

PROVIDER: S-EPMC1138006 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Project description: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.

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Differential inactivation of mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine.

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