Identification of the uvrA gene product

Abstract

The proteins synthesized by the plasmid pDR2000, a recombinant plasmid with the Escherichia coli uvrA and ssb ( lexC) genes cloned on pBR322, were examined using the maxicell procedure developed by Sancar et al. (1979). The proteins synthesized by this plasmid include those expected from pBR322 plus two others, a small protein ( M r = 18,500) and a large one ( M r = 114,000). The molecular weight and DNA binding properties of the 18,500 M r protein indicate that it is the product of the ssb gene. Derivatives of pDR2000 were prepared in which the uvrA gene was inactivated by insertion of the γδ transposon (Tn1000). In each of these derivatives the 114,000 M r protein was not made, indicating that it is the product of the uvrA gene. By extrapolating from the relative amounts of ssb and uvrA proteins synthesized in maxicells, we estimate that a normal cell contains only about 20 uvrA polypeptide chains. The uvrA protein binds to both single and double-stranded DNA. A procedure is described for purifying uvrA protein to radiochemical homogeneity, and subsequent gel filtration of this isolated native uvrA protein shows that it is a monomer. When γδ is inserted into the uvrA gene, truncated uvrA polypeptides resulting from premature termination at nonsense codons in the inserted sequence are observed. From the sizes of these truncated polypeptides and the sites of the insertions determined by restriction mapping, the direction of transcription of the uvrA gene was determined to be counter-clockwise on the E. coli genetic map.