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.     

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.     

RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12

Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells. Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme. We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant. The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV. The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled. We concluded that respiration shutoff requires RecBC enzyme activity. The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities. We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity. This strain did not shut off its respiration. The presence or absence of other RecBC enzyme activities in this mutant is not known. These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.     

Purification and subunit structure of recBC DNase from Escherichia coli harboring a recB and recC genes-inserted plasmid

recBC DNase of Escherichia coli has been purified from the transformant, HB101/pFS11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography. The purified enzyme was obtained in an overall yield of 3%. The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis. The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125,000 and 135,000, and isoelectric points of 5.6 and 5.7, respectively.     

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.     

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.     

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.     

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.     

Involvement of recB and recC genes of Escherichia coli in precise transposon excision

The mutations texA343 and texA344, which increase transposon excision, are complemented by multicopy plasmids carrying recC+ and recB+, respectively, but not by derivative plasmids in which the recC and recB genes are inactivated. This shows that texA343 and texA344 are mutations in recC and recB, respectively.     

A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19]

The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features. The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage. Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent. The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme. The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells. The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function. No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid.     

Characteristics of Some Multiply Recombination-Deficient Strains of Escherichia coli

Strains of Escherichia coli have been made carrying lesions in more than one gene determining recombination. The following genotypes were constructed and verified: recC22 recB21 recA(+), recC22 recB21 recA13, recC22 recB(+)recA13, and recC(+)recB21 recA13. All multiple rec(-) strains carrying recA13 were similar to AB2463, which carries recA13 alone, in their UV sensitivities, recombination deficiencies, and inabilities to induce lambda phage in a lysogen. However, whereas AB2463 shows a high rate of ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown, the multiple rec(-) strains showed the low level characteristic of strains carrying recC22 or recB21 alone. The strain carrying both recC22 and recB21 was similar in all properties to the single mutants, suggesting that both gene products act in the same part of the recombination and UV repair pathways. It is concluded that in a Rec(+) strain, the recA(+) product acts to inhibit DNA breakdown determined by the recC(+) and recB(+) products.     

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.     

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.     

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR]. In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.     

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

Synthesis of linear plasmid multimers in Escherichia coli K-12.

Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322. A mutation in sbcB increases the proportion of plasmid DNA as linear multimers. A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis. Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication. Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers. The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.     

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.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...