Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity
- 28 Downloads
UnlikeEscherichia coli, the closely related bacteriumSalmonella 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 theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. 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 mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. 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 ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.
Key wordsEscherichia coli Salmonella typhimurium SOS mutagenesis Chimeric proteins UmuC
Unable to display preview. Download preview PDF.
- Friedberg EC, Walker GC, Siede W (1995) DNA repair and mutagenesis. ASM, Washington, DCGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
- Skavronskaya AG, Stepanova NF, Andreeva IV (1982) UV-mutable hybrids ofSalmonella incorporatingEscherichia coli region adjacent to tryptophan operon. Mol Gen Genet 15:315–318Google Scholar
- Sweasy JB, Witkin EM (1993) Novel SOS phenotypes caused by second-site mutations in therecA430 gene ofEscherichia coli. Biochimie 73:437–448Google Scholar
- Witkin EM, RoegnerManiscalco V, Sweasy JB, McCall JO (1987) Recovery from ultraviolet light-inhibition of DNA synthesis requiresumuDC gene products inrecA718 mutant strains but not inrecA + strains ofEscherichia coli. Proc Natl Acad Sci USA 84:6804–6809Google Scholar