• Corpus ID: 21121512

A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine.

@article{Crone1993ASA,
  title={A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine.},
  author={Tina M. Crone and Anthony E. Pegg},
  journal={Cancer research},
  year={1993},
  volume={53 20},
  pages={
          4750-3
        }
}
Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli ada gene product AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA… 
Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases*
TLDR
Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein.
Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine.
TLDR
Results suggest that the residues arginine-128 and tyrosine-114 are involved in the DNA binding properties of the AGT.
Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6-benzylguanine.
TLDR
It is confirmed that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates.
Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine.
TLDR
The results suggest that the C145A mutant AGT binds to O6-methylguanine lesions in DNA and prevents their repair by NER, and support the hypothesis that mammalian AGTs bind to O4-methylthymine but repair the lesion so slowly that they effectively shield it from more efficient repair byNER.
Specific labeling of O6-alkylguanine-DNA alkyltransferase by reaction with O6-(p-hydroxy[3H]methylbenzyl)guanine.
TLDR
Results demonstrate that AGT accepts HMBG as a substrate and becomes inactivated by transfer of a p-hydroxymethylbenzyl residue to the cysteine-145 acceptor site and indicate that [3H]HMBG is a potentially useful reagent for the detection and localization of AGT activity and for the investigation of its mechanism of action.
Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein
TLDR
The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O6-position of guanine.
Activation of human O6-alkylguanine-DNA alkyltransferase by DNA.
TLDR
Results demonstrate that the alkyltransferase binds to a region of DNA covering at most 12 bases and undergoes a conformational change which facilitates the reaction of adducts at the O6-position of guanine with the cysteine acceptor site on the protein.
...
...

References

SHOWING 1-10 OF 15 REFERENCES
Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.
  • M. DolanR. MoschelA. Pegg
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 1990
TLDR
Results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.
Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanine-DNA alkyltransferase.
TLDR
It is concluded that for efficient AGT depletion, an allyl or benzyl group attached through exocyclic oxygen at position 6 of a 2-aminopurine derivative is required.
Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase
TLDR
It is inferred that there is an endogenous source of O6MeG or O4MeT DNA damage in E. coli that is prevalent in nondividing cells and that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the OGT MTase also protects cells from killing by alkyLating agents.
Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analyses of three residues at or near the methyl acceptor site.
TLDR
To elucidate the significance of a highly conserved amino acid sequence of methyl transferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined.
Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.
TLDR
The results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor and suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA- MGMT complex formation may constitute a significant secondary lesion.
Repair of DNA containing O6‐alkylguanine
  • A. PeggT. Byers
  • Biology, Chemistry
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology
  • 1992
TLDR
Inactivation of theAlkyltransferase sensitizes tumor cells to these chemotherapeutic alkylating agents and may prove a useful therapeutic strategy.
Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts.
TLDR
Antisera were raised in rabbits to three peptides which correspond to sequences of amino acids present in human O6-alkylguanine-DNA alkyltransferase (residues 1-11, 8-20 and 197-207), but this protein was found to be absent from Mer- tumor cell lines.
Effect of O6-benzylguanine on the sensitivity of human tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea and on DNA interstrand cross-link formation.
TLDR
The studies demonstrate that the therapeutic index of BCNU can be increased when given in combination with O6-benzylguanine.
Regulation of repair of alkylation damage in mammalian genomes.
  • S. MitraB. Kaina
  • Biology
    Progress in nucleic acid research and molecular biology
  • 1993
...
...