Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.

Generation of a detailed physical and genetic map of the ilv-metE-udp region of the Escherichia coli chromosome.

The entire ilv-metE-udp region of the Escherichia coli chromosome has been cloned in two steps using the lambda replacement vector EMBL4. A detailed restriction map for approximately 70 X 10(3) bases of DNA has been generated. The gpp and udp structural genes have been identified, the cya and metE genes have been physically located, and the direction of recQ gene transcription has been determined. By examining a variety of plasmid subclones, 44 polypeptides have been detected using maxicell and minicell analysis, accounting for 70% of the maximum coding capacity of the entire region. On the basis of the observed gene density in the ilv-metE-udp region, a total number of 3000 genes is predicted for the entire E. coli chromosome. In addition, anomalies in cotransduction frequencies that have been observed in this region have been interpreted by employing a new formula that incorporates the effects of different transducing fragment representations and recombination probabilities.

DNA repair in Escherichia coli: identification of the uvrD gene product.

A 2.9-kilobase (kb) Pvu II DNA fragment that contains the uvrD gene of Escherichia coli K-12 has been cloned in both low-copy and multiple-copy plasmid vehicles. The low-copy uvrD plasmid (pVMK49) complements a variety of uvrD, uvrE, and recL mutations. In contrast, the same strains carrying the 2.9-kb fragment in a multiple-copy plasmid (pVMK45) remain sensitive to ultraviolet light (UV). Additionally, pVMK45 transformants of wild-type E. coli are sensitive to UV and methyl methanesulfonate and appear to be recombination deficient. The cloned uvrD gene does not complement the dominant uvrD3 allele. The 2.9-kb Pvu II insert in these plasmids encodes a single 76,000-dalton protein, which, on the basis of insertional inactivation experiments with the Tn1000 transposon, must be the uvrD gene product. These data confirm earlier genetic analysis which suggested that recL, uvrE, and uvrD were all allelic. The direction of transcription of the uvrD gene has also been determined.

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.

Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.

Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins.