Identification of the uvrD gene product of Salmonella typhimurium LT2

Abstract

The product of the uvrD gene of Salmonella typhimurium LT2 and Escherichia coli K-12 is thought to play a role in both the correction of mismatched bases and the repair of DNA damage, since insertion mutations in the uvrD gene increase the spontaneous mutation frequency and make the cells more sensitive to killing by UV irradiation. To clone the uvrD gene of S. typhimurium, we first generated a uvrD-specific probe by using DNA from an S. typhimurium uvrD421::Tn5 mutant. This probe was used to screen a lambda library of S. typhimurium DNA. Bacteriophage carrying intact uvrD+ genes were subsequently identified, and the uvrD+ gene was subcloned onto a low-copy-number vector. By using a combination of Tn1000 insertion mutagenesis and the maxicell technique, the product of the uvrD gene was shown to be a 75,000-dalton protein, and the relative direction of transcription of this protein was determined. Introduction of a low-copy-number plasmid carrying the S. typhimurium uvrD+ gene into uvrD insertion mutants of either S. typhimurium or E. coli restored the spontaneous mutation frequency and degree of UV sensitivity to the levels in the corresponding uvrD+ strains.