recBC DNase Research Articles

ATP-stimulated polymerase activity involving DNA polymerase I and a recB-dependent factor in extracts of Escherichia coli cells.

ATP-stimulated DNA polymerase activity involving DNA polymerase I has been found to be present in cell extracts from wild type and recC mutant strains of Escherichia coli, but not in extracts from recB strain. The activity has been separated from recBC DNase by DEAE-cellulose ion exchange. It is suggested that recB-dependent factor is involved in the ATP-stimulation of polymerase. Evidence is provided that this stimulation may be due to the interaction of recB-dependent factor with DNA polymerase I.

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.

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins.

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.

RecF-dependent induction of recA synthesis by coumermycin, a specific inhibitor of the B subunit of DNA gyrase.

Genetic and biochemical studies on enzymes known to be involved in regulating DNA supercoiling yield a complex spectrum of effects on the Escherichia coli SOS system. Previous studies indicated that only inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the E. coli SOS system. Here we show that coumermycin, an antibiotic that acts on the DNA gyrase B subunit, can also induce. Like nalidixic acid induction, coumermycin induction is dependent on the recBC DNase. In both cases induction apparently results from a response of the cell to the DNA gyrase-inhibitor complex rather than just the loss of DNA gyrase activity. However, unlike induction by the DNA gyrase A-specific antibiotics, coumermycin induction also requires the recF gene product. This demonstrates a functional relationship between DNA gyrase and the recF gene product.

Fine structure of the [formula omitted] and [formula omitted] gene region of [formula omitted

For genetic and enzymatic study of recBC DNase, the Escherichia coli thyA, recC, recB and argA gene region cloned in a cosmid was subcloned into the pBR322 plasmid vector and its fine restriction map was made. Complementation analysis showed that these genes were located in a 19kb Bam HI fragment in the order thyA recC recB argA , so far as estimated. The recA recB recC cells harboring plasmid subcloned with this Bam HI fragment exhibited 12.3-fold increase in recBC DNase activity. Clones carrying the recB and the recC gene expressed recB and recC function, respectively, and the initiation sites of transcription of these genes were detected by S1 nuclease mapping, proving that the recB and recC genes consist of independent cistrons.

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

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

Identification of the Escherichia coli recB and recC gene products.

Functional recB and recC genes are required for normal conjugative genetic recombination in Escherichia coli1–3 and mutations in either of these genes lead to deficiency in an ATP-dependent nuclease known as the RecBC DNase or exonuclease V (refs 4–6). Widely different molecular weights have been reported for the putative subunits of this enzyme7,8 and genetic and biochemical experiments have left some doubt about whether or not recB and recC are different genes. We have now cloned the recB and recC genes separately into the multiple copy number plasmid pAT153 and identified the gene products by transposon inactivation and gel electrophoresis. The products of the recB and recC genes are proteins of molecular weights (MW) 135,000 and 125,000, respectively.

Transcription regulation of colicin Ib synthesis.

Bacteriophage Mud (Casadaban and Cohen 1979) was used to bring the transcription of the gene for beta galactosidase (lacZ) under the control of the promoter of the structural gene for colicin Ib (cia(Ib)) on a derivative of the Col plasmid Col-Ib.P9. Transcription of this fusion operon was stimulated by agents which damaged cellular DNA (mitomycin C, bleomycin and colicin E2). Increased transcription of the cia-lacZ operon could be detected within 13 min of the addition of these agents. In a strain bearing the tif-1 (recA441) mutation, constitutive expression of the SOS DNA repair system at 42 degree C also increased transcription of the cia-lacZ operon. Transcription of the cia-lacZ operon was also stimulated by inhibition of DNA gyrase activity with nalidixic acid but not with novobiocin. Transitory inhibition of protein synthesis with chloramphenicol or by proline starvation of a proline auxotroph did not stimulate cia-lacZ transcription. Transcription of the cia-lacZ operon was substantially reduced in the presence of a recA mutation, but was largely unaffected by a mutation in recB affecting the RecBC DNase or by catabolite repression. Control experiments in which the production of colicin Ib was measured confirmed that the experiments with the fusion operon gave an accurate indication as to the activity of the wild type cia gene except for the effect of catabolite repression, where we observed up to 99% reduction in colicin Ib production in strains carrying mutant crp or cya alleles. The overall results confirm previous suggestions that there was considerable similarity between the regulatory systems controlling production of colicins and the repressor-dependent regulation of lambdoid prophage induction.

Partial purification and properties of an exonuclease inhibitor induced by bacteriophage Mu-1.

From an induced lysogen of bacteriophage Mu-1, we partially purified a substance of high molecular weight that blocks the action of several exonucleases on double-stranded DNA. The presence of the inhibitor in cell-free extracts is dependent on induction of a Mu prophage. The Mu-related inhibitor acts by binding to double-stranded DNA rather than by interacting with the DNase. The inhibitor protects linear duplex DNA of Mu, P22, and phi X174am3 from exonucleolytic degradation by recBC DNase and lambda exonuclease. Single-stranded DNA, however, is not protected by the inhibitor from degradation by either recBC DNase or exonuclease I. The inhibitor preparation contains a protein that binds to linear duplex DNA, but not to circular duplex DNA; ends are required for binding to occur. Single-stranded DNA is not a substrate for the binding protein. These and other results suggest that the binding protein and the inhibitor are the same activity.

Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase.

RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate).

4] Assay for type II restriction endonucleases using the Escherichia coli rec BC DNase and duplex circular DNA

Publisher Summary This chapter deals with assay for type II restriction endonucleases using the Eseherichia coli recBC DNase and duplex circular DNA. Duplex, circular DNA is not a substrate for the recBC DNase of Escherichia coli (exonuclease V); however, linear DNA is digested by the enzyme to acid-soluble nucleotides. Therefore, the conversion of duplex, circular DNA to linear DNA by a restriction endonuclease in the presence of an excess of recBC nuclease results in the production of acid-soluble nucleotide in amounts proportional to the endonuclease activity. Properties of the assay are further described in the chapter. An estimation of endonuclease activity by this assay gave excellent quantitative agreement with results obtained by agarose gel dectrophoresis assays when either EcoR1 or Alul restriction endonucleases were utilized. EcoR1 breaks at one site on colicin E1 DNA and makes cohesive ends, whereas the Alu 1 enzyme makes roughly 50 nonstaggered breaks with this DNA. These results indicate that the exonuclease is present in excess so that a DNA molecule is essentially digested completely into acid-soluble fragments immediately upon receiving one double-stranded restriction cleavage.

19] Preparation and properties of the HpaI and HpaII endonucleases

Publisher Summary This chapter discusses the preparation and properties of the HpaI and HpaII endonucleases. Two restriction endonuclease activities, HpaI and HpaII are isolated from Haemophilus parainfluenzae. Endonuclease HpaI has been isolated in homogeneous form, while HpaII has been purified free of contaminating nuclease and phosphatase activities. HpaI recognizes the DNA sequence and cleaves the phosphodiester bonds in both strands as indicated. HpaII recognizes the DNA sequence and cleaves the phosphodiester bonds in a staggered fashion. Assay method is further explained. Two assays have been used during the purification of HpaI and HpaII. The first assay is qualitative and involves the cleavage of γ DNA by the respective enzyme into fragments of discrete size. These fragments are separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Characteristic sets of fragments are seen for each of the enzymes. HpaI cleaves γ DNA 11 times while HpaII cleaves γ, DNA more than 50 times. The second assay is quantitative and measures the conversion of closed circular RF fd DNA, which contains a single HpaI site, to the linear form which is sensitive to degradation by RecBC DNase.

Mechanisms of Action of the Type-I Restriction Endonuclease, EcoB, and the recBC DNase from Escherichia coli

Mechanisms of Action of the Type-I Restriction Endonuclease, EcoB, and the recBC DNase from Escherichia coli

Inactivation of phage repressor in a permeable cell system: role of recBC DNase in induction.

UV light causes inactivation of phage (phi80) repressor molecules in a plasmolyzed, permeable cell preparation of Escherichia coli. Induction without UV irradiation occurs when the permeable cells are incubated in the presence of four deoxyribonucleoside triphosphates and ATP. The induction triggered by dNTP's requires a functional recBC gene product and is associated with degradation of the DNA replication fork. The role of recBC DNase in the induction of prophage and SOS functions in general is discussed.

Early events and mechanisms in the induction of bacterial SOS functions: analysis of the phage repressor inactivation process in vivo.

Different inducing agents and treatments produced distinctly different kinetic patterns of inactivation of prophage repressor molecules. The different patterns were related to differences in the initial altered states of DNA that were produced. The timing of appearance of DNA degradation was correlated with the time needed for repressor inactivation. These characteristics suggest that all the inducing treatments lead to the formation of a final predegradative DNA structure(s) (probably involving scissions) that is acted on by specific DNases, including the recBC DNase, to produce the signals for the induction of prophage.

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.

Integration of the λ genome in the absence of site-specific integration function: Isolation of an abnormally permuted prophage λ

The mechanism of integration of λ bio ll, which is deleted of all the known λ recombination genes, was studied using bio deleted hosts as recipients. The presence of rec BC DNase and exo I in the recipient cells affected the fate of λ bio ll DNA. In nine of ten immλ + transductants, insertion of the λ bio ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.

Selective inhibition of the dnase activity of the recBC enzyme by the DNA binding protein from Escherichia coli.

In the presence of the Escherichia coli DNA binding protein, single-stranded DNA is resistant to both the endo- and exonucleolytic activities of the recBC DNase. Linear duplex DNA, on the other hand, is unwound at a normal rate, but converted to large, single-stranded fragments which are resistant to further hydrolysis. Therefore, in the presence of the binding protein and linear duplex DNA, the recBC enzyme acts not as a DNase, but primarily as an ATP-dependent unwinding enzyme, able to generate large, single-stranded material. Duplex circular DNA containing short, single-stranded gaps is also resistant to the hydrolysis in the presence of the binding protein.

Postinfection control by bacteriophage T4 of Escherichia coli recBC nuclease activity.

Infection by bacteriophage T4 has previously been shown to cause a rapid inhibition of the host recBC DNase, an ATP-dependent DNase that is required for genetic recombination in Escherichia coli. We report here the partial purification of a protein ("T4 rec inhibitor") from extracts of T4-infected cells and some characteristics of the in vitro inhibition reaction with purified inhibitor and recBC nuclease. This inhibitory activity could not be purified from extracts of uninfected E. coli. Both the ATP-dependent exonuclease and DNA-dependent ATPase activities of recBC DNase are inhibited by T4 rec inhibitor. Experiments suggest that the inhibitor interacts with the nuclease in a stoichiometric manner. The biological significance of this inhibition is discussed with respect to control reactions in phage-infected cells.