The
            <i>recBC</i>
            Deoxyribonuclease of
            <i>Escherichia coli:</i>
            Isolation and Characterization of the Subunit Proteins and Reconstitution of the Enzyme

Lieberman, Robert P.; Oishi, Michio

doi:10.1073/pnas.71.12.4816

Cited by 60 publications

(27 citation statements)

References 13 publications

(7 reference statements)

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“…Amundsen et al (in press) have demonstrated that this gene encodes a third subunit of the RecBC enzyme that is required for the exonuclease activity of ExoV. The possibility of a third subunit had been suggested previously by the in vitro reconstitution experiments of Lieberman and Oishi (27) and by studies of purified ExoV by Dykstra et al (12). Our mutations affecting plasmid maintenance and the mutations described by Amundsen et al (in press) almost certainly affect the same gene.…”

Section: Discussionmentioning

confidence: 70%

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli

Biek¹,

Cohen²

1986

J Bacteriol

202

120

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We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element TnlO into recD resulted in increased concatemerization and loss of pSC101 and ColEl-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coi chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.The maintenance of plasmids as extrachromosomal elements in bacterial cells is dependent on a number of complex processes. Of primary importance is the requirement for plasmid replication, which commonly involves at least some host-encoded replication functions (see Scott [39] for a review). Replication of the pSC101 plasmid (7) requires the Escherichia coli dnaA gene product (21). Mutations in other genes involved in chromosomal replication, including dnaB, dnaC, and dnaG (14,22), also affect replication of this plasmid. Stable maintenance of pSC101 in growing cell populations also requires a plasmid locus (named par for partitioning) that is involved in the distribution of plasmid molecules to daughter cells at the time of cell division (29,45). Earlier work has shown that the pSC101 par region does not encode a protein but instead includes partition-related segments (30) that appear to be necessary for plasmids in the intracellular pool to be counted and partitioned as individual molecules (45). To better understand the mechanism by which pSC101 segregates during cell division, we undertook to identify and characterize E. coli genes encoding products involved in the plasmid maintenance (Pma) phenotype.We report here the isolation and characterization of mutations that affect the stable maintenance of pSC101, as well as that of certain other plasmids. We present evidence that these mutations, which can alter plasmid stability by increasing the frequency of multimer formation and are located in a previously unidentified E. coli gene, affect the activity of exonuclease V (ExoV). MATERIALS AND METHODSStrains, media, and general methodology. Relevant bacterial genotypes, plasmids, and phages are listed in Table 1. The media used for these studies have been previously described (31). Minimal medium was M9 or M63 supplemented with 0.2% glucose (or another carbon source), 1 ,ug of thiamine per ml, and any required L-amino acids (Sigma Chemical Co.) at 40 ,ug/ml. Solid media contained 1.5% agar (Difco Laboratories) or 0.7% agar for soft agar overlays. Lactose indicator plates contained 40 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside; Boehringer Mannheim) per ml in minimal medium containing...

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Section: Discussionmentioning

confidence: 70%

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli

Biek¹,

Cohen²

1986

J Bacteriol

202

120

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“…Note that all of these phenotypes result from defects in processes involving free DNA ends, which act as the entry points for the RecBCD enzyme. The importance of the RecD subunit was only fully appreciated when it was identified as being a physical component of the "RecBC" holoenzyme required for the exonuclease activity (7,37,105,178). This late discovery was, in part, due to the complexity of interpreting the recD phenotype.…”

Section: Discoverymentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

501

599

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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“…ExoV activity is missing in extracts from either recB or recC mutants. However, one subunit required for ExoV activity, named ax by Lieberman and Oishi, is not eliminated by mutations in either gene (21). Chaudhury and Smith (5) found nuclease-deficient mutations in the vicinity of the recBC genes, and it was later shown that the ExoV activity can be eliminated by mutations in recD while leaving the bacteria proficient for recombination (1,4,5).…”

Section: Methodsmentioning

confidence: 99%

“…The apparent identity of the restriction maps of these plasmids with those of the plasmids used for the initial linear transformation demonstrated that the chromosome had not suffered any gross deletions in this region as a result of allele transfer to the chromosome. DISCUSSION The recB, recC, and recD genes of E. coli code for three polypeptides that make up a single complex protein (1, 21; reviewed in reference 10) containing several enzymatic activities, including a helicase (39) and ExoV (21). In wildtype E. coli, the RecBCD complex promotes homologous recombination, and mutations in recB or recC result in decreased recombination activity and increased sensitivity to UV light.…”

Section: Methodsmentioning

confidence: 99%

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

1989

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Wild-type Escherichia coil are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.In contrast to some other bacteria, Escherichia coli is not readily transformable by linear pieces of DNA (34). This is due in part to degradation of the incoming DNA by intracellular exonucleases (13,43). Oishi and Cosloy (8,9,26) showed that E. coli strains lacking exonuclease V (ExoV) by virtue of recB or recC mutations can be transformed by linear DNA if they carry as well the sbcB (and sbcC) mutations that restore recombination proficiency to the recB recC mutant. Similarly, recD mutants also are lacking in exonuclease activity, but unlike the recB recC mutants are robust and recombination proficient without the requirement of additional mutations (5).Jasin and Schimmel (14) and Winans et al. (44) used recB recC sbcB strains to transform with linearized plasmids and homologously replace a chromosomal allele with an allele that had been modified. Yamaguchi and Tomizawa (46) and Gutterson and Koshland (12) used strains defective in polA to transfer genes from plasmids to the chromosome. Strains carrying recB, recC, and sbcB grow poorly and generally carry a mutation in sbcC (22). In addition, strains carrying polA are not robust (15). These considerations make the general use of such strains unattractive.Using the E. coli chemotaxis system genes as a chromosomal target, we show here that recD strains can be transformed with linear DNA. The availability of recD alleles that are mutant by virtue of a mini-Tet insertion facilitates the construction of transformable derivatives of strains of interest by one-step bacteriophage-mediated transduction.MATERIALS AND METHODS Strains, media, and chemicals. The bacterial strains, plasmids, and phages used are listed in Tables 1 and 2. LB medium and Tryptone broth were as described previously (2). The lactose phenotypes of the bacterial strains were determined on agar plates containing 5-bromo-4-chloro-3-indoyl-p-D-galactopyranoside (X-Gal) at 20 ,ug/liter and isopropyl-3-D-thiogalactopyranoside (IPTG) at 1 mM. Methyl esterase assays. The activity of the CheB protein, a methyl esterase whose activity is modulated in response to attractants and repellents, can be measured in vivo by the amount of radioactive methanol released from cells that have been incubated with methyl-labeled radioactive methionine (16,18,19,37,41,42). Steady-state cumulative evolution of methanol was measured by the method of Toews and Adler (41) as described by Kehry et al. (16). Stimulus-dependent esterase activity was measured in the flow apparatus as described by Kehry et al. (6,16,17).Transfer of chromosomal DNA to plasmids. The method for transferring chromosomal DNA to plasmids is ...

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The recBC Deoxyribonuclease of Escherichia coli: Isolation and Characterization of the Subunit Proteins and Reconstitution of the Enzyme

Abstract: After dissociation of the E. coli recBC DNase (ATP-dependent DNase) with concentrated NaCl, two subunit proteins were isolated by ion exchange chromatography. Combination and subsequent incubation of the subunits resulted in the appearance of the original DNase.

Cited by 60 publications

References 13 publications

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

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