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PubMed1987

Kinetics of recB-dependent repair: relationship to post-UV inactivation of the prophage.

By making use of the temperature-sensitive mutant recB270, we showed that the RecBCD enzyme is needed for repair between 1 and 4 h after UV exposure. recB-dependent prophage inactivation (Petranović et al. (1984), Mol. Gen. Genet., 196, 167-169) takes place in all dying cells during the same period of time. The kinetics of decrease in the yield of recombinants in phage-propage crosses resemble those of prophage inactivation in UV-irradiated bacteria. This indicates that recombination processes (including site-specific recombination required for prophage excision) are blocked in cells destined to die. On the basis of our results, we suggest that a large fraction of damaged cells is rescued by the RecA-RecBCD recombination pathway. If repair is unsuccessful, RecA-RecBCD recombination intermediates persist in the irradiated cells leading to prophage inactivation.

Coliphages↗
PubMed1995

Evaluation of Escherichia coli recBC sbcBC mutants for cloning by recombination in vivo.

In vivo recombination as a tool for plasmid construction was analyzed using a model system based on the properties of the RecF pathway in Escherichia coli. This pathway was used after a double strand break (DSB) effected by restriction enzyme cleavage of the plasmids of interest. DSB repair was shown to be independent of the methylation state of the vector or insert DNA, of the type of restriction enzyme used (5' or 3' overhanging or blunt ends) and of dephosphorylation of the vector and/or template. Since the E. coli repair system does not recognize insertions, this recombination mechanism can be used to exchange cDNAs between different vectors. Some implications of the results on the mechanism of recombination are discussed.

Bacterial Proteins↗
PubMed1991

Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway.

Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed (mut H,L,S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10(3)-fold. The interspecific recombination in mutS or mutL mutants is only approximately 10-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased approximately 10(3)-fold in a recC mutant.

Conjugation, Genetic↗
PubMed1991

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.

Chromosome Mapping↗
PubMed1991

RecBC promoted repair of bleomycin damage in Escherichia coli.

The repair response of Escherichia coli K-12 to bleomycin was examined in Rec- mutants showing differential sensitivity to this agent. Sedimentation analysis of the cellular DNA showed incision after bleomycin treatment. The subsequent reformation of the DNA, found in the wild-type and the recD mutant, was abolished in the recB and delayed in the recF and recBC sbcB mutants. The bleomycin-induced SOS response was reduced in strains containing recB or recBC sbsB mutations. It is suggested that the RecBCD pathway has the main role in the efficient repair of bleomycin-induced DNA damage.

Bleomycin↗
PubMed1991

Interaction of lambda Gam protein with the RecD subunit of RecBCD enzyme increases radioresistance of the wild-type Escherichia coli.

By making use of the gam(+)-plasmid, the so-called gam-dependent radioresistance was studied. This resistance is the result of the interaction between Gam protein (encoded by the gam gene of lambda) and RecBCD enzyme of Escherichia coli. gam-dependent radioresistance is observed in recB+ recC+ recD+ but not in recB+ recC+ recD- cells. It is suggested that Gam protein interacts specifically with the RecD subunit of RecBCD enzyme; the RecBC complex probably retains its activity in the presence of this viral protein.

Bacteriophage lambda↗
PubMedDate not supplied

Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair.

The recD, recJ and recN genes of Escherichia coli K-12 have been shown to be involved in genetic recombination and DNA repair in this organism. Yet, mutation of any one of these genes does not seem to interfere much with the recovery of recombinants from conjugational crosses. Strains carrying all possible combinations of mutations inactivating these genes were constructed and examined for their recombination proficiency and sensitivity to UV light. The recD recJ and recJ recN double mutants are moderately sensitive to UV light and slightly deficient in recombination. A combination of mutations in all 3 genes produced strains that are very deficient in recombination (50- to 100-fold reduction) and strikingly sensitive to UV light. We conclude that these genes provide overlapping activities that compensate for one another in the single mutants. On the basis of these and other data, recombination genes are classified into 3 epistatic groups that define activities which function pre-synaptically or post-synaptically to promote genetic exchanges catalysed by RecA.

Bacterial Proteins↗
PubMed1992

Aphidicolin inhibits DNA polymerizing activity but not nucleolytic activity of Escherichia coli DNA polymerase II.

We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide. Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases. Aphidicolin had no detectable effect on the 3'----5' exonuclease activity. The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA. This mode of action is the same as that on eukaryotic DNA polymerase alpha. The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.

Aphidicolin↗
PubMed1993

Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities.

rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.

DNA Helicases↗
PubMed1983

Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease.

Small polydisperse circular (spc) DNAs of mouse thymocytes were purified by a procedure involving nitrocellulose column chromatography and the treatment of ATP-dependent DNase, which acts only upon linear DNA molecules. Nitrocellulose column chromatography prior to the enzyme treatment was essential because digestion of linear DNA duplexes by the enzyme was inhibited by the presence of concomitant single-stranded DNAs. Mitochondrial DNAs were eliminated by linearization with XhoI and digestion with ATP-dependent DNase. The size distribution of the purified spc DNA molecules ranged from 0.2 micron to more than 28 micron, with a mean length of 5.4 micron. Circular molecules of more than 0.4 micron long (or 1.2 kb) were free from the contamination of linear DNA fragments and pure enough to be cloned into plasmids.

Animals↗
PubMed1986

Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda.

The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene). A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed. Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages. The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability.

Bacteriophage lambda↗
PubMed1986

Host/vector interactions which affect the viability of recombinant phage lambda clones.

A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells. We report experiments which address the mechanism of this inhibition. The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells. In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function. Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities. These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes. This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation. Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.

Bacterial Proteins↗
PubMed1986

Factors which equalize the representation of genome segments in recombinant libraries.

Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec+ hosts. We have found that 9% of phage lambda clones that contain 15-20-kb insertions of human or Drosophila DNA are inhibited on rec+ hosts and as a result will become under-represented in amplified genomic libraries. We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage lambda vectors. This loss may be diminished if the vector carries either a chi element or a functional gam gene. The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD. We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts.

Animals↗
PubMed1986

Localization of the gam gene of bacteriophage mu and characterisation of the gene product.

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification.

Bacterial Proteins↗
PubMed1991

Ultraviolet light-induced plasmid-chromosome recombination in Escherichia coli: the role of recB and recF.

Bacterial host cells of different rec genotypes were used to investigate genetic requirements of ultraviolet light (UV)-induced homologous plasmid-chromosome recombination. Plasmid DNAs which contained a wt or mutant lacY gene were irradiated with UV prior to transformation into Escherichia coli host strains which contained the complementary lacY allele. Surviving transformants were screened to determine the directions of UV-induced recombinational exchange between the bacterial and plasmid lacY genes, by assaying lactose utilization. Nonreciprocal chromosome-to-plasmid recombination was 100% dependent on the recA gene and greater than 80% dependent on the recF gene, but not dependent upon the recB gene of E. coli. In contrast, reciprocal plasmid-chromosome recombination was strictly dependent on the recA gene, greatly dependent (approx. 80%) on the recF gene, and moderately dependent on the recB gene. Nonreciprocal plasmid-to-chromosome recombination was only induced at very low frequencies, and appeared to be moderately dependent on the recB gene, but not dependent on the recF gene. UV-induced plasmid-chromosome recombination appeared to proceed by a two-step mechanism. In this model, the initial step is recF-dependent, recB-independent, and either resolves to become a nonreciprocal chromosome-to-plasmid recombinant, or proceeds to the second step. The second step is moderately recB-dependent and results in the reciprocal exchange of plasmid-chromosome sequences.

Bacterial Proteins↗
PubMed1991

A general structure for DNA-dependent DNA polymerases.

In addition to the general 3'-5' exonuclease domain described by Bernad et al. [Cell 59 (1989) 219-228] significant amino acid (aa) sequence similarity has been found in the C-terminal portion of 27 DNA-dependent DNA polymerases belonging to the two main superfamilies: (i) Escherichia coli DNA polymerase I (PolI)-like prokaryotic DNA polymerases, and (ii) DNA polymerase alpha-like prokaryotic and eukaryotic (viral and cellular) DNA polymerases. The six most conserved C-terminal regions, spanning approx. 340 aa, are located in the same linear arrangement and contain highly conserved motifs and critical residues involved in the polymerization function. According to the three-dimensional model of PolIk (Klenow fragment), these six conserved regions are located in the proposed polymerization domain, forming the metal and dNTP binding sites and the cleft for holding the DNA template. Site-directed mutagenesis in the phi 29 DNA polymerase supports some of these structural predictions. Therefore, it is likely that a 'Klenow-like core', containing the DNA polymerase and 3'-5' exonuclease activities, has evolved from a common ancestor, giving rise to the present-day prokaryotic and eukaryotic DNA polymerases.

Amino Acid Sequence↗
PubMed1991

Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits.

The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB. RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.

Adenosine Triphosphate↗