Effect of mutations in recB, recC, sbcB and recF genes controlling the homologous recombination in Escherichia coli cells on transposition of Tn1

The mutations in the genes controlling the homologous DNA recombination in Escherichia coli cells effect the efficiency of Tn1 transposition. Mutations in recB and recC genes decrease 50-fold the frequencies of Tn1 transposition. Introduction of an additional mutation in sbcB gene increase transposition frequency for three orders as compared with the one registered in wild type cells. Inactivation of sbcB gene in the wild type cells does not affect transposition significantly. Mutation in recF gene results in the great decrease of transposition when it is introduced into multiple recBC sbcB mutant, but not into the wild type bacteria. The possibility of two pathways for Tn1 transposition existing in Escherichia coli cells is discussed, as well as possibility of existence of similar stages in transposition and recombination controlled by the same genes.     

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.     

Properties of the recB and recC gene products of Escherichia coli

The properties of the recB and recC gene products of Escherichia coli were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase (recBC enzyme) but showed apparent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products encoded by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton proteins, respectively. These results suggest that the recB and recC genes are the structural genes of the beta and alpha subunits, respectively, of the recBC enzyme. A gene that encodes a protein of about 100,000 daltons was found to be located between the recB and recC genes. But the product of this gene was shown not to be included in the recBC enzyme.     

Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12.

A 19-kilobase BamHI fragment encoding the recB (exonuclease V), recC (exonuclease V), ptr (protease III), thyA, and argA genes of Escherichia coli K-12 was cloned into a multicopy plasmid (pCDK3). In E. coli maxicells, the plasmid specified the synthesis of seven polypeptides of 140,000 (recC), 128,000 (recB), 110,000 (ptr), 53,000 (argA), 50,000, 33,000 (thyA), and 22,000 Mr, as well as beta-lactamase and chloramphenicol acetyltransferase. From analysis of subclones and Tn1000 insertions, it appears that the 110,000- and 50,000-Mr proteins originated from the ptr DNA coding sequence which is located between the recB and recC genes. Although recC, ptr, and recB were physically closely linked and transcribed in the same direction, they do not appear to constitute an operon. Cells carrying pCDK3 contained a 30- to 50-fold increase in exonuclease V activity, without affecting cell viability.     

Quantitation of the involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-ray-induced DNA double-strand breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.     

Lethality of rep recB and rep recC double mutants of Escherichia coli

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the λ Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The reasons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBts recCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC -dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.     

The dependence of postreplication repair on uvrB in a recF mutant of Escherichia coli K-12.

Summary Mutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated vir and wild type rates of excision gap closure in repairing UV damage to their own DNA. The same mutants showed reduced rates of postreplication repair strand joining. When uvrA ^- recF^- or uvrB ^- recF^- strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m². We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF may be similar. An intermediate in postreplication repair may accumulate in the uvr ^- recF^- strain.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Construction of recombinant lambda phages that carry the E. coli recB and recC genes

Summary A fragment of the E. coli chromosome including the recC gene has been cloned by in vitro recombinant DNA techniques into a phage vector to give the recombinant phage drecC. This was used to derive the phage drecBC by in vivo recombination. On lysogenisation of recB and recC mutants with drecBC wild type levels of UV-resistance and RecBC DNase activity were restored. Infection of E. coli with drecBC led to the synthesis of phage-coded proteins of 125 kilodaltons (kd) and 135 kd that were not synthesised on infection with the original vector, whereas a 125 kd protein but not a 135 kd protein was synthesised in similar experiments with drecC. The recombinant phages, which are unable to form plaques, presumably due to the deletion of essential phage genes during their construction, provide useful starting points from which to subclone the recB, recC, and the neighbouring thyA and argA genes individually into multiple copy plasmid vectors.     

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.     

ColE1 plasmid mutants affecting growth of an Escherichia coli recB recC sbcB mutant.

The level of cyclic GMP was less than one molecule per organism in dormant, germinated, and outgrowing spores of Bacillus megaterium. A significant level (approximately 8 pmol/g, dry weight) of cyclic GMP was found in early to mid-log phase cells, but the level fell to below 0.2 pmol/g, dry weight, in late-log phase and only rose slightly to approximately 0.9 pmol/g, dry weight, in stationary phare. No significant amount of cyclic GMP was detected in the growth medium at any time.     

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.     

Chromosome Transfer from F-lac+ Strains of Escherichia coli K-12 Mutant at recA, recB, or recC

The frequency of chromosome transfer from various recombination-deficient F-lac(+) donor strains was estimated by standardizing the yield of conjugants receiving a male chromosomal marker against the level of episome transfer in the mating mixture. The efficiency of chromosome transfer from newly formed F-lac(+) cells carrying recB21 or recC22 was more than 50% of the wild-type value, although it was about 10 and 20%, respectively, if the male cell lines had become established. In contrast, recA13 donors transmitted the chromosome with less than 10(-4) of the normal frequency. If chromosome transfer from F-lac(+) strains reflects the cutting and subsequent joining of homologous single strands of episomal and chromosomal deoxyribonucleic acid by recombination, these results imply that the completed unions are not made in recA cells, but can be effected with more than 50% of normal efficiency in newly formed partial diploids mutant at either recB or recC. Thus, the defective stage in recA mutants may precede strand joining, whereas the deficiency in recB or recC cells may involve a later step in recombinant formation.     

Protein Z is not an endonuclease of the recF recombination pathway in Escherichia coli K12

A 74 kD protein was extracted from Escherichia coli cells and purified under the physiological conditions. The protein is able to catalyze the reactions of endonucleolytic degradation of plasmid DNA. The genetic determinant coding for the 74 KD protein synthesis has been localized between 17 and 27 min on Escherichia coli chromosomal map. The endonuclease previously described as a recF gene dependent "protein Z" (Krivonogov S. V., Novitskaja V. A. Mol. Gen. Genet., 1982, v, 187, p. 302) is shown to be independent of the integrity of Escherichia coli recF gene.     

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.     

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.     

Inhibition of postreplication recombinational repair by DNA-gyrase antagonists in Escherichia coli

In the present study we investigated the possible involvement of DNA-Gyrase in postreplication repair in E. Coli. It was observed that nalidixic acid and oxolinic acid (which are known-antagonists of DNA-Gyrase) inhibited recombinational repair. These results strongly suggest that the nicking closing activity of DNA-Gyrase is essential for efficient recombinational repair.