title: Isolation and characterization of novel plasmid-encoded umuC mutants.
abstract: Most inducible mutagenesis in Escherichia coli is dependent upon the activity of the UmuDC proteins. The role of UmuC in this process is poorly understood, possibly because of the limited number of genetically characterized umuC mutants. To better understand the function of the UmuC protein in mutagenic DNA repair, we have isolated several novel plasmid-encoded umuC mutants. A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitro by exposure to hydroxylamine. Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promote umuC-dependent spontaneous mutator activity were identified by a colorimetric papillation assay. Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremely useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.
authors.affiliation: Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
authors.forename: R
authors.initials: R
authors.lastname: Woodgate
authors.forename: M
authors.initials: M
authors.lastname: Singh
authors.forename: O I
authors.initials: OI
authors.lastname: Kulaeva
authors.forename: E G
authors.initials: EG
authors.lastname: Frank
authors.forename: A S
authors.initials: AS
authors.lastname: Levine
authors.forename: W H
authors.initials: WH
authors.lastname: Koch
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013014:SOS Response, Genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D004259:DNA-Directed DNA Polymerase