Analysis of chimeric UmuC proteins: identification of regions in Salmonella typhimurium UmuC important for mutagenic activity.
Unlike Escherichia coli, the closely related bacterium Salmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of the S. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins of E. coli and S. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostly E. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostly S. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein of S. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of the S. typhimurium UmuC protein into the UmuC protein of E. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212-395 and 396-422 of E. coli UmuC with those from S. typhimurium resulted in reduced mutability, while substitution of residues 26-59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability of S. typhimurium can be attributed to mutations located within residues 26-59 of the S. typhimurium UmuC protein.
Koch
W H
WH
Molecular Biology Branch, Food and Drug Administration, Washington, DC 20204, USA.
Kopsidas
G
G
Meffle
B
B
Levine
A S
AS
Woodgate
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C078202:UmuC protein, E coli; D004259:DNA-Directed DNA Polymerase