N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli: multiple roles for UmuC protein.
Backmutations of an ochre (UAA) nonsense defect in the tyrA gene of Escherichia coli were induced by N-ethyl-N-nitrosourea (ENU) in both UmuC+ and UmuC- strains. This site is particularly flexible to base substitution mutation and all but one (TAA-->TGA) substitution can be recovered using a reversion assay. Employing direct sequencing of polymerase chain reaction-amplified genomic DNA and/or colony-hybridization methods, the changes induced by several separate doses of ENU in a total of 587 independent backmutations were investigated. In the UmuC+ strain, all possible single-base substitutions were recovered. Different frequencies for individual single-base substitutions were obtained and correlations with the surrounding base sequence could be seen. Transitions occurred most frequently at thymine residues having a purine on the 5'-side while transversions occurred more frequently at thymine residues having a cytosine on the 5'-side. In the UmuC- strain, ENU failed to induce A:T-->T:A and A:T-->C:G transversions and the frequency of A:T-->G:C transitions was reduced. However, an unidentified class of extragenic mutations were induced to a greater extent. These results suggest distinct pathways for ENU-induced mutagenesis at ethylated thymine residues and delineate several separate functions for the UmuC protein.
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Department of Microbiology, Southern Illinois University, Carbondale 62901.
D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005038:Ethylnitrosourea / Q000494:pharmacology*; D016296:Mutagenesis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D004259:DNA-Directed DNA Polymerase; D005038:Ethylnitrosourea