Document Detail


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.
MedLine Citation:
PMID:  9934856     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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, O4-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.
Authors:
S Edara; S Kanugula; A E Pegg
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Carcinogenesis     Volume:  20     ISSN:  0143-3334     ISO Abbreviation:  Carcinogenesis     Publication Date:  1999 Jan 
Date Detail:
Created Date:  1999-02-18     Completed Date:  1999-02-18     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8008055     Medline TA:  Carcinogenesis     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  103-8     Citation Subset:  IM    
Affiliation:
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution
Codon / genetics
DNA Damage
DNA Repair
Escherichia coli / drug effects*,  genetics
Guanine / analogs & derivatives,  chemistry
Humans
Methylnitronitrosoguanidine / toxicity*
Mutagenesis*
O(6)-Methylguanine-DNA Methyltransferase / genetics,  physiology*
Point Mutation
Recombinant Fusion Proteins / physiology
Thymidine / analogs & derivatives,  chemistry
Grant Support
ID/Acronym/Agency:
CA-18137/CA/NCI NIH HHS; CA-57725/CA/NCI NIH HHS; CA-71976/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Codon; 0/Recombinant Fusion Proteins; 20535-83-5/O-(6)-methylguanine; 50-89-5/Thymidine; 50591-13-4/O-methylthymidine; 70-25-7/Methylnitronitrosoguanidine; 73-40-5/Guanine; EC 2.1.1.63/O(6)-Methylguanine-DNA Methyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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