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

Suvarchala Edara, Sreenivas Kanugula, Anthony Pegg

30 Scopus citations

Abstract

Human O6-alkylguanine-DNA alkyltransferase (AGT) counteracts the mutagenic and toxic effects of methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by removing the methyl group from O6-methylguanine lesions in DNA. The methyl group is transferred to a cysteine acceptor residue in the AGT protein, which is located at residue 145. The C145A mutant of AGT in which this cysteine is converted to an alanine residue is therefore inactive. When this C145A mutant was expressed in an Escherichia coli strain lacking endogenous alkyltransferase activity, the number of G:C→A:T mutations actually increased and the toxicity of the MNNG treatment was enhanced. These effects were not seen when an E. coli strain also lacking nucleotide excision repair (NER) was used. The enhancement of mutagenesis and toxicity of MNNG produced by the C145A mutant AGT was not seen with another inactive mutant Y114E that contains a mutation preventing DNA binding, and the double mutant C145A/Y114E was also ineffective. These results suggest that the C145A mutant AGT binds to O6-methylguanine lesions in DNA and prevents their repair by NER. The inactive C145A mutant AGT also increased the number of A:T→G:C transition mutations in MNNG-treated cells. These mutations are likely to arise from the minor methylation product, O6-methylthymine. However, expression of wild-type AGT also increased the incidence of these mutations. These results 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 by NER.

Original languageEnglish (US)
Pages (from-to)103-108
Number of pages6
JournalCarcinogenesis
Volume20
Issue number1
DOIs
Publication statusPublished - Jan 23 1999

All Science Journal Classification (ASJC) codes

  • Cancer Research

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