UvrD Protein Research Articles

An in vitro complementation assay for the Escherichia coli uvrD gene product.

An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.

Transcription of the uvrD gene of Escherichia coli is controlled by the lexA repressor and by attenuation.

The nucleotide sequence of the control region and the presumptive N-terminal portion of the uvrD gene of Escherichia coli K-12 has been determined. The 1190 base pairs of DNA examined include the likely coding sequence for the first 258 amino acids of the uvrD protein. The transcription promoter for the uvrD gene was identified upstream of the protein coding region. Synthesis of messenger RNA in vitro from this promoter was inhibited by purified lexA protein. The lexA protein was found to bind downstream from the promoter at a sequence, CTGTATATATACCCAG, which is homologous to other known lexA protein binding sites. In the absence of the lexA protein, approximately half of the messages initiated in vitro at the uvrD promoter terminate after about 60 nucleotides at a sequence which resembles a rho-independent terminator. These results indicate that the uvrD gene is induced during the SOS response, and that the expression of the gene may also be regulated by transcription attenuation.

The uvrD gene of E. coli encodes a DNA-dependent ATPase.

Mutations in the uvrD gene of Escherichia coli K-12 result in multiple phenotypes, including increased sensitivity to UV light and ionizing radiation, decreased ability for host cell reactivation, increased spontaneous mutation frequency and decreased rate of genetic recombination1–6. It has been shown, moreover, that double mutants having mutations in both uvrD and polA genes are conditionally lethal7–10. The pleiotropic phenotypes of the uvrD mutants suggest that the uvrD gene product may have important roles in the processes of DNA repair, recombination and replication. To study the function of the uvrD gene, we have constructed hybrid phages and plasmids that carry the gene11. Here we have analysed the proteins synthesized by the hybrid phages and report that the uvrD gene product is a polypeptide of molecular weight (MW) 75,000. We present evidence that the uvrD protein possesses a DNA-dependent ATPase activity.