Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.     

Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli.

The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC). Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9. Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids. In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability. Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control. Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E. coli.     

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.     

Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.     

Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5'' leads to 3'' exonuclease of DNA polymerase I.

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.     

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.     

Genetic Analysis of Recombination-Deficient Mutants of Escherichia coli K-12 Carrying rec Mutations Cotransducible with thyA

The rec mutations carried by 20 strains of Escherichia coli K-12 which are defective in genetic recombination and sensitive to ultraviolet light and X rays, and whose lambda lysogens show spontaneous phage production, have been mapped near thyA. In 15 of the strains, the rec mutation fails to complement recB21 but complements rec-22. The other five strains carry a rec mutation which complements recB21 but not rec-22. These mutations map closer to thyA than those which fail to complement recB21. They therefore appear to be defective in a different recombination gene, denoted recC. The order of recB and recC on the linkage map of E. coli K-12 is thyA-recC-recB-argA.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

rorA mutation of Escherichia coli K-12 affects the recB subunit of exonuclease V.

The ATP-dependent nuclease, exonuclease V, of Escherichia coli plays an important role in repair and recombination. The enzyme is composed of two subunits, one of which is the product of the recB and recC genes. In this communication it is shown by mapping and complementation experiments that the rorA mutation, which results in radiation sensitivity but not the loss of recombination ability, is an allele of the recB gene.     

Involvement of the recB-recC Nuclease (Exonuclease V) in the Process of X-Ray-Induced Deoxyribonucleic Acid Degradation in Radiosensitive Strains of Escherichia coli K-12

The ras, polA, exrA, recA, and uvrD3 strains of Escherichia coli K-12 degrade their deoxyribonucleic acid more extensively than wild-type strains after X irradiation. The relationship of the recB-recC nuclease (exonuclease V) to the degradation process in these strains was determined by comparing the degradation response of the original strains with that of strains containing an additional recB21 or recC22 mutation. The initial rate of degradation in ras, polA12, exrA, and recA13 strains after an exposure of 20 to 30 kR was reduced more than 10-fold by the presence of an additional recB21 or recC22 mutation. The extent of degradation in these irradiated strains after 90 to 120 min of incubation was reduced two- to fivefold. In the uvrD3 strain, a recC22 mutation caused a fourfold decrease in initial degradation rate and reduced the extent of degradation after 90 min of incubation by a factor of 1.6. The results are consistent with the statement that the degradation process is normally dependent on exonuclease V activity. However, the observation that 10 to 30% degradation always occurred even in recB or recC strains, which lack this enzyme, suggests that alternative degradation mechanisms exist.     

Identification and genetic mapping of the structural gene for an essential Escherichia coli membrane protein.

Attempts to isolate conditionally lethal recB and recC mutations of Escherichia coli K-12 by P1 localized mutagenesis led to the identification of the structural gene for an essential membrane protein. Located on a 1.5-kilobase-pair DNA fragment which physically mapped immediately 5' to the thyA gene, the product of the umpA (unidentified membrane protein) gene is a 25,000 Mr membrane-associated polypeptide. These results provide an explanation for why several research groups have been unable to obtain chromosomal deletions of the entire thyA gene. A possible interaction between the umpA and thyA genes is also discussed.     

Escherichia coli recBC deletion mutants.

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.     

Differential Thermolability of Exonuclease and Endonuclease Activities of the recBC Nuclease Isolated from Thermosensitive recB and recC Mutants

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.     

Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes

Biochemical evidence is presented that confirms exonuclease V of Escherichia coli consists of three distinct subunits encoded by the recB, recC, and recD genes. The recD gene encodes a Mr 60,000 polypeptide and physically maps 3' to the recB structural gene. The role of the recD subunit in exonuclease V function has been examined by comparing the catalytic activities of the purified RecBCD enzyme with the RecBC enzyme. The RecBC enzyme retains significant levels of DNA-dependent ATPase activity and DNA helicase activity. Endonucleolytic activity on single-stranded covalently closed DNA becomes ATP-dependent. Exonucleolytic activity on either single- and double-stranded DNA was not detected. Taken together with the phenotypic properties of recD null mutants, it appears that the exonucleolytic activities of the RecBCD enzyme are not required for genetic recombination and the repair of either UV-induced photoproducts or mitomycin C-generated DNA cross-links, but are essential for the repair of methyl methanesulfonate-induced methylation.     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

Genetic Analysis of Mutations Indirectly Suppressing recB and recC Mutations

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.     

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.