Role of exonucleases V and VIII in adenosine 5''-triphosphate- and deoxynucleotide triphosphate-dependent strand break repair in toluenized Escherichia coli cells treated with X-rays.

The repair of X-ray-induced strand breaks was studied in permeabilized Escherichia coli recBC cells deficient for the adenosine 5'-triphosphate (ATP)-dependent exonuclease V and in recBC sbcA cells that possess the ATP-independent exonuclease VIII. It is shown that repair induced by additon of ATP does not take place in recBC and recBC sbcB cells and is limited in recBC sbcA cells. ATP-dependent repair is nevertheless observable if together with ATP a mixture of deoxynucleotide monophosphates is supplied to the cells. These data fit with the assumption that in wild-type cells ATP-dependent repair involves exonuclease V-induced deoxyribonucleic acid degradation and rephosphorylation of the degradation products which are reused for deoxyribonucleic acid polymerase I-dependent break closure. Repair in the presence of deoxynucleotide triphosphates rejoins a similar fraction of breaks in all strains tested irrespective of the amount of postirradiation degradation resulting from exonuclease V and exonuclease VIII activities. Thus, exonuclease V is dispensable for deoxynucleotide triphosphate-dependent repair, i.e., does not "clean" the ends of breaks produced by X-irradiation. ATP- and deoxynucleotide triphosphate-dependent repair are not additive and seem to repair the same population of deoxyribonucleic acid molecules damaged by X-irradiation.     

Differential Thermolability of Exonuclease and Endonuclease Activities of the recBC Nuclease Isolated from Thermosensitive recB and recC Mutants

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.     

Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts

It is shown that circular PM2 DNA with two gaps of 13 nucleotides per molecule is degraded by purified recBC enzyme from Escherichia coli to acid-soluble material at a rate which is less than one tenth of the rate of solubilization of linear duplex DNA. Increasing the gap length in the circular DNA to 40-650 nucleotides does not affect the breakdown of the molecules by the recBC enzyme, nor does it change the proportions of the products formed (acid-soluble material, acid-insoluble fragments and non-degraded molecules). On the other hand, terminal gaps in linear duplex DNA produced by limited digestion with either exonuclease III or lambda exonuclease significantly reduce the rate of the degradation by the recBC enzyme, particularly when the gaps exceed 100 nucleotides. The results suggest that the recBC enzyme does not cleave gaps in circular DNA at random positions, but possibly at the junction between single-stranded and duplex DNA or close to it. The degradation of gapped circular DNA by purified recBC enzyme was used to search for an inhibitor of the recBC enzyme in extracts from ultraviolet-irradiated cells. No such inhibitor has been observed but rather a weak stimulatory factor for the solubilization of gapped circular DNA by the recBC enzyme. Thus, the experimental system appears not to be suited as a test in vitro for an ultraviolet-induced inhibitor of the recBC enzyme which has been postulated to be produced in recA+ lexA+ cells of E. coli after ultraviolet irradiation.     

Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions.

The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.     

Cisplatin induces DNA double-strand break formation in Escherichia coli dam mutants

Escherichia coli dam cells are more susceptible to the cytotoxic action of cisplatin than wildtype. Dam mutS or dam mutL bacteria, however, are resistant to this agent indicating that active mismatch repair sensitizes dam cells to cisplatin toxicity. Genetic data, obtained previously, were consistent with the generation and repair of cisplatin-induced double-strand breaks (DSBs). We measured DSB formation in temperature-sensitive dam recB mutants, after exposure to cisplatin, using pulse field gel electrophoresis and observed an increase in linear 100-300 kb DNA fragments corresponding to approximately 15-45 double strand breaks per genome. The formation of these DSBs was temperature and dose-dependent and was decreased in recBC bacteria at the permissive temperature or in dam(+) or mutS control strains. There was a three-fold increase in circa 2 mb linear chromosomal fragments in dam recBC strains at the non-permissive temperature compared to recBC alone. We show that dam priA strains are not viable suggesting that DSB formation is dependent on DNA replication restart. The sensitivity of priA mutants to cisplatin is also consistent with this conclusion.     

SP02 particles mediating transduction of a plasmid containing SP02 cohesive ends

SP02 particles that mediate transduction of plasmid pPL1010, a 4.6-megadalton derivative of pUB110 containing an Eco RI endonuclease-generated fragment of SP02 deoxyribonucleic acid that spans the cohesive ends, exhibit three unusual features: the transducing particles have a lower buoyant density than infectious particles; the transduction of pPL1010 occurs at high efficiency; and the transducing activity of the particles is relatively resistant to ultraviolet irradiation when the recipient is recombination proficient. Evidence is presented which indicates that SP02(pPL1010) particles carry the plasmid predominantly as a linear multimer having a molecular mass comparable to that of infectious SP02 deoxyribonucleic acid (ca. 31 megadaltons). The plasmid monomers in the linear multimer appear oriented in the same polarity. The buoyant density difference between infectious and transducing particles appears to be due mainly to the buoyant density difference between pPL1010 (1.699 g/cm3) and SP02 deoxyribonucleic acid (1.702 gm/cm3).     

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.     

Granulosis Virus Deoxyribonucleic Acid: a Closed, Double-Stranded Molecule

Studies on deoxyribonucleic acid purified from the granulosis virus of Trichoplusia ni have revealed the presence of a closed, double-stranded superhelix which sediments at 95S relative to relaxed circles (74S) and linear (60S) forms. Molecular weight estimates show that this insect virus deoxyribonucleic acid has a size of 100 x 10(6) daltons.     

A CHROMATOGRAPHIC STUDY OF HYDROLYSIS IN THE FEULGEN NUCLEAL REACTION

In a study on Feulgen hydrolysis of frozen-dried alcohol-fixed lily anthers, a chromatographic technique was developed to analyze the acid hydrolysate for some of the degradation products of nucleic acid. Hydrolysis was accomplished by 10 per cent perchloric acid at 20°C., and a typical hydrolysis time-Feulgen intensity curve was obtained, with maximum staining occurring at 19 hours. Microphotometric measurements indicated that the amount of stain per nucleus was no different from amount in nuclei fixed and hydrolyzed by more conventional procedures. Uracil-containing material (from ribonucleic acid) was almost completely separated from thymine-containing material (deoxyribonucleic acid) of tissue sections by acid treatment for 1½ hours. Adenine (purines), as the base, was effectively all removed from the deoxyribonucleic acid at the time of optimum hydrolysis. Detectable amounts of thymine-containing material appeared in the hydrolysate shortly after the onset of hydrolysis; and the amount increased rapidly with increased hydrolysis time. At the time of optimum hydrolysis approximately two-thirds of the total deoxyribonucleic acid thymine was lost. The removal of these thymine-containing fragments was linear with respect to time during the first 24 hours and occurred at a relatively high rate. Removal after 24 hours was also linear but was at a markedly lower rate. These results would suggest that two kinds of deoxyribonucleic acid exist in lily anthers; an acid-labile fraction amounting to approximately three-fourths of the total, and an acid-resistant fraction making up the remainder. In the Feulgen procedure much of the labile fraction is lost by the time of optimum hydrolysis and is not stained; most of the stable fraction remains in the tissue and is stained. In light of these findings the use of the Feulgen method as a means of determining cytochemically relative amounts of deoxyribonucleic acid in nuclei by measuring their Feulgen dye content was discussed.     

The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Summary The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec ⁺, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric from (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells.On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell.The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.     

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).     

Comparison of the requirements for ribonucleic acid synthesis with the requirements for deoxyribonucleic acid synthesis in animal tissues

Ribonucleic acid polymerase and deoxyribonucleic acid polymerase have been partially purified from bovine lymphosarcoma, lymph node, and thymus. An examination of the deoxyribonucleic acid requirements of the two enzymes indicates that “native” deoxyribonucleic acid is the preferred template for ribonucleic acid synthesis; heat-denatured deoxyribonucleic acid is considerably less active. The primer requirements for deoxyribonucleic acid synthesis differ: “native” deoxyribonucleic acid is usually inactive, while denatured deoxyribonucleic acid is active. The two enzymes also differ in pH optima and in their requirements for metal cofactors.     

Role of Recbc Function in Formation of Chromosomal Rearrangements: A Two-Step Model for Recombination

The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.     

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.     

Fast genome editing in Bacillus subtilis

Bacillus subtilis is a model organism for Gram‐positive bacteria and widely used in the study of cellular functions and processes including protein secretion, sporulation, and signal transduction. It is also an important industrial host for the production of proteins and chemicals. Generally, genome editing of B. subtilis often needs the construction of integration vectors in Escherichia coli, linearizing the constructed plasmids, and subsequent transformation of the linear deoxyribonucleic acid via natural competence or electroporation. In this work, we examined the feasibility to directly transform and integrate B. subtilis using linear deoxyribonucleic acid from Gibson assembly without the need for cloning in E. coli. Linear deoxyribonucleic acid of 8–10 kb showed the highest transformation efficiency which was similar to that of using linearized plasmids constructed in E. coli. This method shortens the overall process from 1 week to 1 day and allows the integration of multiple genes in one step, providing a simple and fast method for genome editing in B. subtilis.     

Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.

The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120).     

Orientation Dependence in Homologous Recombination

Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs.     

Template requirement of maize RNA polymerase

Maize RNA polymerase utilizes heated deoxyribonucleic acid more effectively than native deoxyribonucleic acid as a template for ribonucleic acid synthesis. A ribonucleic acid-deoxyribonucleic acid hybrid accumulates in the presence of heated deoxyribonucleic acid. The amount of product formed with either native or heat-denatured deoxyribonucleic acid does not exceed the amount of deoxyribonucleic acid added as template.     

sbcB15 And DeltasbcB mutations activate two types of recf recombination pathways in Escherichia coli

Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and DeltasbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and DeltasbcB mutations on DNA repair after UV and gamma irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than DeltasbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as DeltasbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC DeltasbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.