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PubMed1983

Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8.

An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.

Adenosine Triphosphate↗
PubMed1985

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.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Old and new concepts for the role of chi in bacterial recombination.

The DNA sequence 5'[GCTGGTGG]3', which is called chi, stimulates recombination that is mediated by the RecBCD pathway of Escherichia coli. In 1981, a model was proposed in which the RecBCD enzyme enters DNA at a double-chain end. The enzyme then travels between the chains by unwinding and rewinding the DNA at different rates so that the traveling enzyme becomes encumbered by a region of unwound DNA. Upon meeting chi, the enzyme was supposed to cut one of the two unwound chains, generating thereby a recombinagenic single-chain end. The model, based on microscopical observations of RecBCD enzyme interacting with linear duplex DNA, was supported by the subsequent finding that RecBCD acting in vitro under certain conditions did deliver a nick at chi. This widely embraced model has been challenged by a model in which the exonuclease activity of RecBCD destroys DNA from the enzyme's entry site to chi. The role of chi according to the new model is to inhibit this nuclease activity of RecBCD, perhaps by ejecting the RecD subunit from the enzyme, thereby revealing the enzyme's recombinase activity.

Base Sequence↗
PubMed1995

The amino acid sequence required for 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase.

We studied the 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase by site-directed mutagenesis. Among seven mutants constructed, two mutant DNA polymerases with an amino acid substitution of Gly184 --> Asp or Gly192 --> Asp were confirmed to be deficient in this exonuclease. The two positions corresponded to those of the Escherichia coli DNA polymerase I mutants defective in 5' --> 3' exonuclease, polA480ex and polA214. These results provide experimental support for the proposed amino acid sequence essential for the 5' --> 3' exonuclease activity associated with eubacterial polymerase I-like DNA polymerases (family A), including E.coli and Thermus aquaticus.

Bacillus↗
PubMed1996

Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC1 (regulator of chromosome condensation) and virus DNA remains linear.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5 degrees C, a nonpermissive temperature. In this mutant at 39.5 degrees C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5 degrees C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5 degrees C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5 degrees C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

Animals↗
PubMed1999

Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells.

Autographa californica nucleopolyhedrovirus (AcMNPV) DNA polymerase was purified from virus-infected cells using conventional chromatographic methods. The enzymatic activity of fractions eluting from single-stranded agarose gels was found to exactly coincide with a single polypeptide with an apparent molecular mass of approximately 110,000 Da on denaturing polyacrylamide gels stained with Coomassie blue. This purification scheme resulted in a 228-fold purification of AcMNPV DNA polymerase with recovery of 3.5% of the initial activity. The specific activity of the most purified fraction of DNA polymerase was 5000 units/mg, which is sufficiently high to eliminate the possibility that contaminants significantly contribute to the polymerase activity. Preparations of purified DNA polymerase had 3'-5' exonuclease activity, but no 5'-3' exonuclease activity. The proofreading activity was apparently an intrinsic property of the enzyme as the ratio of nuclease activity to polymerase activity was constant throughout purification. Using a singly-primed M13 DNA template, RF-II DNA was detected within 3 min, indicating a polymerization rate of 40 nt/s. The effects of several DNA polymerase inhibitors on the enzymatic activity of purified DNA polymerase were also determined.

Animals↗
PubMed1984

Changes in ATP concentration in Escherichia coli during induction of the SOS system by mitomycin C and bleomycin.

Treatment of Escherichia coli with bleomycin induced a dramatic increase in ATP concentration in the first 30 min. Afterwards, in RecA+ strains, ATP dropped quickly to values similar to those of untreated cells. Mutants of E. coli defective in either RecA protein or RecA protease activity did not show this decrease, indicating that it was due to the action of RecA protease. The increase in ATP in the first 30 min was dependent on RecBC exonuclease activity and must have been due to substrate level phosphorylation, since an uncoupler such as dinitrophenol did not affect it. Nevertheless, mitomycin C did not induce any change in ATP pools of RecA+ strains, at least during 120 min following treatment. The implications of these findings are discussed in relation to the possible pathways of activation of RecA protease.

Adenosine Triphosphate↗
PubMed1987

Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme.

Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.

Conjugation, Genetic↗
PubMed1992

Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme.

In Escherichia coli, constituents of the main recombination pathway are provided by the genes recA (RecA protein) and recBCD (RecBCD enzyme). Recombination in conjugation experiments and repair of UV damage of E. coli mutants deleted for recA, for recBCD or for recA plus recBCD were restored, although to different degrees, by the cloned recA and recBCD genes from Serratia marcescens or Proteus mirabilis. When both recombination enzymes were from the same species, repair and recombination efficiencies had the order E. coli greater than S. marcescens greater than P. mirabilis. However, the P. mirabilis recA plus recBCD genes resulted in higher levels of repair and recombination than those obtained with one component from P. mirabilis (recA or recBCD) and the other from E. coli or S. marcescens. The data provide evidence for the similarity of RecABCD pathways of recombination among enteric bacteria and suggest an in vivo advantage of an intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme over interspecies combinations. This could point to a cooperation between these basic recombination enzymes. The molecular processes which could be involved are discussed.

Cloning, Molecular↗
PubMed1993

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli.

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

ATP-Dependent Proteases↗
PubMed1989

4-quinolones and the SOS response.

The SOS DNA repair system is induced in bacteria treated with 4-quinolones. However, whether the response exacerbates or repairs the damage caused by these drugs is still unclear. The recA13 and the recB21 mutations impair recombination repair and render bacteria unable to induce the SOS response when treated with nalidixic acid or other agents that affect DNA synthesis. However, UV treatment induces the SOS response in recB21 mutants but not in recA13 mutants. Both these mutants are hypersensitive to nalidixic acid and, therefore, either recombination repair or SOS repair would appear to repair DNA damage caused by the drug. However, since the lexA3 mutation (which also renders bacteria incapable of inducing the SOS response without affecting recombination repair) had no effect on the susceptibility of bacteria to nalidixic acid, the SOS response neither contributes to nor repairs DNA damage caused by the drug. Consequently, it would seem that the hypersensitivity of the recA13 and recB21 mutants to nalidixic acid is due to their deficiency in recombination repair. This view was confirmed by testing a recA430 mutant that is recombination-repair proficient but SOS repair-deficient and finding it to be no more sensitive to nalidixic acid than its parent. Thus it would appear that, although induced by nalidixic acid treatment, the SOS DNA repair system does not play any role in bacterial responses to the damage caused by the drug. In contrast, the recombination repair system does repair damage caused by nalidixic acid.

Anti-Infective Agents↗
PubMed1999

Characterization of the recD gene of Neisseria gonorrhoeae MS11 and the effect of recD inactivation on pilin variation and DNA transformation.

Pilin antigenic variation in Neisseria gonorrhoeae may result following intrachromosomal recombination between homologous pil genes. Despite extensive study, recA is the only previously characterized gene known to be involved in this process. In this study, the gonococcal recD gene, encoding one subunit of the putative RecBCD holoenzyme, was characterized and its role in pilin variation assessed. The complete recD gene of N. gonorrhoeae MS11 was cloned and its nucleotide sequence determined. The gonococcal recD gene complemented a defined Escherichia coli recD mutant, based on plaque formation of bacteriophage lambda and the restoration of ATP-dependent nuclease activity. Inactivation of the gonococcal recD gene had no measurable effect on cell viability or survival following UV exposure, but did decrease the frequency of DNA transformation approximately threefold. The frequency at which non-parental pilin phenotypes were spawned was 12-fold greater in MS11 recD mutants compared with the parental MS11 rec+ strain. Similar results were obtained using recD mutants that were not competent for DNA transformation. Complementation of the MS11 recD mutant with a wild-type recD gene copy restored the frequency of pilin phenotypic variation to approximately wild-type levels. The nucleotide changes at pilE in the recD mutants were confined to the variable regions of the gene and were similar to changes previously attributed to gene conversion.

Amino Acid Sequence↗
PubMed1997

The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme.

Homologous recombination in Escherichia coli is stimulated at DNA sequences known as chi sites. Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' --> 5' exonuclease. Upon recognition of a properly oriented chi site, the 3' --> 5' exonuclease activity is attenuated. Here we show that in addition to attenuation of the 3' --> 5' exonuclease activity, recognition of chi by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' --> 3'. These results demonstrate that chi is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' --> 5' nuclease activity, and up-regulation of the 5' --> 3' nuclease activity. The consequence of chi recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the chi sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E. coli, and in other organisms such as the yeast Saccharomyces cerevisiae.

Binding Sites↗
PubMed1999

Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo.

DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically.

Bacterial Proteins↗
PubMed1999

Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication.

To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage lambda chromosomes, repressed or not for lambda gene activities. Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends. We report that the RecBC recombinational pathway of E. coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction.

DNA Repair↗
PubMed1999

Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits.

We report here an unusual mechanism for enzyme regulation: the disassembly of all three subunits of RecBCD enzyme after its interaction with a Chi recombination hot spot. The enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein. We show that after reaction with DNA bearing Chi sites, RecBCD enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis. This Chi-mediated inactivation and disassembly of purified RecBCD enzyme can account for the previously reported Chi-dependent loss of Chi activity in E. coli cells containing broken DNA. Our results support a model of recombination in which Chi regulates one RecBCD enzyme molecule to make a single recombinational exchange ('one enzyme-one exchange' hypothesis).

Adenosine Triphosphate↗
PubMed1999

The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation.

Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as chi sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3'-terminal, chi-containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3'-terminal DNA strand, with no requirement for chi. This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with chi.

Blotting, Southern↗