Mutants of bacteriophage λ defective in vegetative genetic recombination

Echolas, H; Gingery, R. E.

doi:10.1016/0022-2836(68)90249-0

Cited by 118 publications

(34 citation statements)

References 26 publications

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“…Lambdologists soon isolated phage mutants that could not recombine, thereby defining the Red pathway, which requires the exo and beta gene products (36,87). exo was shown to encode an exonuclease (79) slightly before the discovery of the RecBCD nuclease.…”

Section: A Brief History Of Recombination In Escherichia Coli and Phagementioning

confidence: 99%

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Smith

2012

Microbiol Mol Biol Rev

155

191

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SUMMARY The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli , the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular ( in vivo ) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.

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Section: A Brief History Of Recombination In Escherichia Coli and Phagementioning

confidence: 99%

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Smith

2012

Microbiol Mol Biol Rev

155

191

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“…In addition, the Abc function of P22, but not the Gam function of X, was found to inhibit recombination in a wild-type host; however, both Abc and Gam inhibited recombination in a recF mutant host. These observations are interpreted as indicating that the recombination systems of both phages, as well as the RecBCD-modulating functions Abc and Gam, all activate the RecF recombination pathway of E. coli.Bacteriophages A and P22 encode their own homologous recombination systems, which promote recombination between phage chromosomes even in the absence of hostencoded RecA and RecBCD proteins (2,4,5,(13)(14)(15) In the experiments summarized in Table 1, wild-type and mutant strains of Escherichia coli bearing plasmids that express various and P22 genes were tested for their abilities to serve as recipients in Hfr-mediated conjugation. In the construction of these plasmids, described previously (5, 11, 12), phage genes were fused to the lacUVS promoter and ligated into the lacI-bearing, tetracycline resistanceconferring vector pMC7.…”

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confidence: 99%

“…Bacteriophages A and P22 encode their own homologous recombination systems, which promote recombination between phage chromosomes even in the absence of hostencoded RecA and RecBCD proteins (2,4,5,(13)(14)(15). This observation raises the question of whether the phage systems can promote other recombination events.…”

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Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions

Poteete

Volkert

1988

J Bacteriol

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Escherichia coli strains bearing wild-type and mutant alleles of various recombination genes, as well as plasmids that express recombination-related genes of bacteriophages A and P22, were tested for their proficiency as recipients in Hfr-mediated conjugation. It was found that the homologous recombination systems of both phages could promote recombination in a recB recC mutant host. In addition, the Abc function of P22, but not the Gam function of X, was found to inhibit recombination in a wild-type host; however, both Abc and Gam inhibited recombination in a recF mutant host. These observations are interpreted as indicating that the recombination systems of both phages, as well as the RecBCD-modulating functions Abc and Gam, all activate the RecF recombination pathway of E. coli.Bacteriophages A and P22 encode their own homologous recombination systems, which promote recombination between phage chromosomes even in the absence of hostencoded RecA and RecBCD proteins (2,4,5,(13)(14)(15) In the experiments summarized in Table 1, wild-type and mutant strains of Escherichia coli bearing plasmids that express various and P22 genes were tested for their abilities to serve as recipients in Hfr-mediated conjugation. In the construction of these plasmids, described previously (5, 11, 12), phage genes were fused to the lacUVS promoter and ligated into the lacI-bearing, tetracycline resistanceconferring vector pMC7. buffer, glucose (0.4%), Casamino Acid hydrolysate (0.2%), thiamine (0.2 ,ug/ml), and streptomycin (100 ,ug/ml). Donor titers were determined by mixing cells with the appropriate volume of L broth, incubating in parallel with the mating mixtures, and plating on L agar. Recombinant titers were determined by plating the mating mixtures on minimal E glucose agar plates containing streptomycin, arginine, histidine, and proline (100 ,ug/ml each). In experiment 2, the donor was MV1955 (a thr-35::Tn9 derivative of BW113). Donor titers were determined as in the other experiments except that the plating medium was L agar containing 25 jig of chloramphenicol per ml. Recipient titers were determined by plating the mating mixtures on L agar containing 100 ,ug of streptomycin per ml. Recombinant titers were determined by plating the mating mixtures on L agar containing both chloramphenicol and streptomycin. In all experiments, titers for control mixtures that lacked either donors or recipients were determined under selective conditions; the background was undetectable in all cases (data not shown).

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“…Recombinationdeficient mutants of bacteriophage and bacteria have been useful in the study of recombination processes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Recombination deficient, radiation-sensitive mutants of Bacillus subtilis have been isolated and described by several investigators (11)(12)(13)(14).…”

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Fate of Transforming DNA After Uptake by Competent Bacillus subtilis : Failure of Donor DNA to Replicate in a Recombination-Deficient Recipient

Davidoff-Abelson¹,

Dubnau²

1971

Proc. Natl. Acad. Sci. U.S.A.

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The fate of radioactively-labeled transforming DNA was studied in a recombination-deficient strain of Bacillus subtilis that carried the recB2 mutation (Rec-) Bacterial transformation is an advantageous system for the study of recombination, since the fate of purified donor DNA can be studied in the course of the different steps before, during, and after integration. Recombinationdeficient mutants of bacteriophage and bacteria have been useful in the study of recombination processes (1-10). Recombination deficient, radiation-sensitive mutants of Bacillus subtilis have been isolated and described by several investigators (11)(12)(13)(14). This paper reports the results of experiments in which the fate of transforming DNA was examined in one of these mutants (recB2). Our results indicate that in recB2, the donor-recipient complex is formed to the same extent as in an isogenic Rec+ strain, but that donor DNA fails to replicate in the recipient after formation of the complex. MATERIALS AND METHODS Bacterial strainsThe bacterial strains used were derivatives of Bacillus subtilis 168. Strains BD170 (thr-5 trp-2) and BD191 (thr-5 trp-2 recB2) were derived from the same parent and were isogenic for all markers except the recB2 marker, which was introduced by transformation as was described (15). The recB2 marker is derived from the mitomycin-sensitive strain mc-1 (12). RecB2 has been shown to be radiation sensitive and deficient in both transformability and transducibility (12)(13)(14). Strain BD204 (hisB2 thy) was used to prepare radioactively-labeled transforming DNA.BD55 (trp-2 hisBW), known also as SB25, was used to assay the biological activity of DNA fractions isolated from the transformed cells. The trp-2 and hisB2 markers are cotransformed at a frequency of about 60%. Media and solutionsThe media for plating and for the development of competence were prepared as described (16). Transformed cells were washed in SP buffer (0.15 M NaCl-0.02 M K2HPO4, pH 7.5). The washed samples were resuspended in lysis medium (0.05 M NaCl-0.1 M EDTA, pH 6.9).SPII medium contained Spizizen salts (17), 0.02% casamino acids, 0.1% Difco yeast extract, 0.5% glucose, 0.5 mM CaC12, 2.5 mM MgCl2, and 50,gg/ml of each required amino acid. Re-extraction experimentsCompetent cells were prepared and frozen as was described (16). Frozen competent Rec+ and Rec-cells were quickly thawed and incubated for 5 min at 370C. To begin the reaction, [3H]DNA, isolated from BD204 as described (16), was then added to a final concentration of 1-2 gg/ml. The specific activity of the DNA preparations ranged from 4 to 6 X 105 cpm/htg. The mixture was incubated at 370C for 7 min or 9 min, at which time an excess (100 ,Ag/ml) of salmonsperm DNA (Calbiochem) was added to stop further DNA uptake. In some experiments the reaction mixture was then transferred to 300C. At different times after addition of the salmon-sperm DNA, samples of the transformed cells were chilled rapidly, washed three times by centrifugation in icecold SP, and resuspended in lysis ...

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Mutants of bacteriophage λ defective in vegetative genetic recombination

Cited by 118 publications

References 26 publications

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions

Fate of Transforming DNA After Uptake by Competent Bacillus subtilis : Failure of Donor DNA to Replicate in a Recombination-Deficient Recipient

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