Purification and Properties of Deoxyribonucleic Acid Binding Factor Isolated from the Surface of Streptococcus sanguis Cells

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S₁ action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.     

Deoxyribonucleases in phytohemagglutinin-stimulated lymphocytes

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by microdisc electrophoresis was used to determine the deoxyribonuclease pattern of human lymphocytes during stimulation with phytohemagglutinin (PHA). Two additional neutral deoxyribonuclease activities are detectable in stimulated cells, one only active with denatured DNA, the other active with native and denatured DNA as substrate, showing a maximum activity after 36 h and increasing in waves respectively. A group of acid deoxyribonuclease activities also shows a maximum after 36 h of stimulation. A neutral deoxyribonuclease active only with native DNA is missing in stimulated lymphocytes. It is suggested that the acid deoxyribonuclease activities and the neutral deoxyribonuclease active only with denatured DNA are involved in DNA synthesis, whereas the involvement of the neutral deoxyribonuclease active with native and denatured DNA in processing of DNA excreted in stimulated lymphocytes is discussed.     

Different deoxyribonucleases in human lymphocytes

The distribution pattern of deoxyribonuclease activities in human lymphocytes has been examined by micro-disc-electrophoresis. Four groups of deoxyribonuclease activities, differing in their electrophoretic mobility, in the nature of their optimal substrate and in their optimal incubation conditions, are characterized. There are two alkaline DNase-activities. One corresponds to DNase I (EC 3.1.4.5), the other having pH optimum of about pH 9.0, prefers denatured DNA as substrate and is not dependent on divalent cations. The fractions with an acid pH optimum can be subdivided into two groups, which differ in their activity towards native DNA, towards denatured DNA, in their activity when succinate is present and in their pH optimum.     

Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected Proteobacteria

SDS-PAGE of cell-free extracts in gels containing bacterial murein or DNA allowed, after enzyme renaturation and staining of nonhydrolysed substrate, the detection of multiple autolysin or deoxyribonuclease activities directly in the gel as zones of clearing. Enzyme profiles of Proteobacteria which are, or were at one time, classified in the genus Pseudomonas were compared. For each species, a relatively large number of autolysin and deoxyribonuclease activities were detected. The distribution, numbers and intensities of zones of clearing in the gel provided complex species-specific patterns. Extensive data from two fundamental, and presumably evolutionarily distinct classes of enzymes were thus generated for purposes of comparison. Neither analysis suggested that these bacteria could represent a single natural cluster of species, lending support to their present multigeneric status. Ethidium-bromide-stained gels could be subsequently stained with Coomassie blue. This allowed the mapping of many deoxyribonuclease activities to particular peptides in the cell-free extract. In addition, modification of the substrate or renaturation buffer enabled a preliminary characterisation of several deoxyribonucleases in terms of their stability, substrate specificity, and other parameters expected to affect enzyme activity. Individual deoxyribonucleases could be located and screened for desired properties without prior purification. Received: 16 December 1996 / Accepted: 20 January 1997     

Activation of DNA primer for DNA polymerase as an ultrasensitive assay for a subgroup of enzymes with deoxyribonuclease activity

Pretreatment of native DNA with nucleases which hydrolyze phosphodiester bonds with liberation of 3′-hydroxyl terminal groups is known to increase the rate of incorporation of nucleotides into DNA by E. coli DNA polymerase I and similar enzymes. Concentration ranges and conditions for this reaction have been established which allow specific detection and quantitative assay of such nucleases. The method permits detection of as little as 0.1 pg of pancreatic deoxyribonuclease, is simple, and requires only commercially available components. It has been utilized to monitor purification of a nuclease from HeLa cell nuclei at concentrations which could not be detected by other methods.     

A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.     

Deoxyribonuclease activity found in Epstein--Barr virus producing lymphoblastoid cells.

A deoxyribonuclease activity from Epstein--Barr (EB) virus producer lymphocyte cell lines which is correlated with viral production and which is not present in virus non-producer or negative lymphocyte cell lines has been purified 220-fold with 20% recovery and characterized. This nuclease copurifies through diethylaminoethylcellulose column chromatography with the EB virus induced deoxyribonucleic acid (DNA) polymerase in EB virus producer cells which was recently reported by this laboratory, but elutes as a separate peak of activity upon phosphocellulose chromatography. This nuclease activity has a sedimentation coefficient of 4.0 S, a strong divalent cation requirement, an alkaline pH optimum, and the ability to utilize both native and denatured lymphocyte DNA as substrate, reducing both to monophosphonucleosides.     

Surface-Bound Nuclease of Staphylococcus aureus: Purification and Properties of the Enzyme

The surface-bound nuclease of Staphylococcus aureus liberated during formation of protoplasts was purified 1,000-fold by chromatography on phosphocellulose. Its properties were compared with those of the known extracellular nuclease, purified 200-fold by the same procedures. The adsorbance of the surface-bound nuclease on phosphocellulose was distinctly different from that of the extracellular nuclease, but other properties of the two enzymes were similar. Both enzymes had a pH optimum of about 10 and required Ca²⁺ for activity. Both enzymes hydrolyzed deoxyribonucleic acid (DNA) and ribonucleic acid, and denatured DNA was a better substrate than native DNA. Both enzymes were inhibited by the same metal ions. Nuclease-less mutants of S. aureus were isolated from S. aureus 209P by using N-methyl-N′-nitroso-N-nitrosoguanidine. These mutants contained neither surface-bound nor extracellular nuclease activity. These results suggest that the surface-bound and extracellular nucleases are expressed from the same cistron of S. aureus.     

The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme.

The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

A conformational basis for the selective action of ara-adenine.

The glycosidic “high anti” conformation is postulated to be the conformation required by the enzymes adenosine kinase and inosine phosphorylase. Purine analogs that are stable in this conformation are either effective substrates or inhibitors of these enzymes. Ara-adenine is shown to be highly unstable in the high anti conformation. The inactivity of ara-adenine as a substrate for both adenosine kinase and inosine phosphorylase is attributed to its inability to assume the high anti conformation specified by these enzymes. That adenosine itself has a local minima in the high anti conformation, as does inosine and guanosine, is required by its ability to inhibit the synthesis of uridylic acid.The minimal cytotoxic properties of ara-adenine is a consequence of its failure, in normal cells, to be converted to the toxic nucleotide form. The ability of ara-adenine to selectively inhibit DNA viruses means that in DNA virus infected cells the conversion of ara-adenine to ara-AMP is facilitated through a mechanism that does not require a substrate high anti conformation.It is apparent that selective antiviral and anticancer nucleoside analogs may be constructed if their conversion to the toxic nucleotide form is prohibited in normal tissues but allowed in cancer cells or virus infected cells. The basis for the selective effects of ara-adenine is that normal cells require a substrate conformation in which ara-adenine is unstable but that certain neoplastic and viral mechanisms for the conversion of ara-adenine to ara-AMP exist which are able to utilize ara-adenine in its stable syn or anti conformations.     

Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.     

Mutants of Diplococcus pneumoniae that Lack Deoxyribonucleases and Other Activities Possibly Pertinent to Genetic Transformation

Mutants of Diplococcus pneumoniae that lacked the two major deoxyribonucleases of the cell—one an endonuclease, the other an exonuclease preferentially active on native deoxyribonucleic acid (DNA)—were obtained. The development of a method for detecting mutant colonies, based on the binding of methyl green to DNA, facilitated isolation of the mutants. Neither enzyme was essential for growth of the cells, for repair of ultraviolet damage, or for any phase of DNA-mediated transformation. Residual deoxyribonuclease activity in the double mutant corresponded to an exonuclease, approximately one-fifth as active as the major exonuclease, that attacked native and denatured DNA equally well. This activity appeared to be associated with the DNA-polymerase enzyme. A mutant that apparently lacked a cell wall lytic enzyme was also fully transformable. A mutant strain that was four times more sensitive to ultraviolet light than the wild type also transformed normally. Recipient cells of this strain were deficient in the repair of ultraviolet-irradiated transforming DNA. Mutants were found which, unlike the wild type, integrated donor markers only with high efficiency, thereby indicating that a particular cellular component that is susceptible to loss by mutation, such as an enzyme, is responsible for low integration efficiency.     

Stimulation of nuclear poly (adenosine diphosphate-ribose) polymerase activity from HeLa cells by endonucleases.

Isolated nuclei from HeLa cells can incorporate labeled ADP-ribose from NAD into an acid-precipitable product, poly(ADP-ribose). This reaction is stimulated by 4-6-fold by the addition of deoxyribonuclease I to the complete reaction mixture. If the nuclei are treated first with deoxyribonuclease I, no effect is seen; the stimulation is only apparent when the two enzymes deoxyribonuclease I and poly(ADP-ribose) polymerase, are operating at the same time. After making several minor modifications in the assay mixture, it was found that another endonuclease, micrococcal nuclease, can also stimulate the poly(ADP-ribose) polymerase activity of HeLa nuclei. A comparison of the two stimulatory effects indicated that the two endonucleases activated to the poly(ADP-ribose) polymerase activity of HeLa nuclei in the same way. Overall this evidence suggests that poly(ADP-ribose) polymerase may have a functional role in the process of DNA repair.     

Mycoplasmal Deoxyribonuclease Activity in Virus-infected L-Cell Cultures

Cell-free extracts of Mycoplasma hominis and medium from 72-hr broth cultures had deoxyribonuclease activity like that of deoxyribonuclease I. Mg(++) stimulated activity, and the pH optimum was between 8.0 and 9.0. Double-stranded or heatdenatured deoxyribonucleic acid (DNA) served as a substrate, and oligonucleotides were produced. Cell-free extracts of L cells infected with M. hominis or M. hominis plus equine abortion virus (equine herpes virus, EAV) had greatly increased activity over that of extracts of L cells or of L cells infected with EAV alone. In the absence of M. hominis, however, extracts had little activity, most of which was in virus-infected cell cultures. Activity was found in the culture medium only in those systems in which M. hominis was present. It is concluded that M. hominis can contribute significant deoxyribonuclease activity to virus-infected as well as virusfree cell cultures. Perhaps the most interesting question arising concerns the ability of EAV, a DNA virus, to replicate successfully despite the presence of deoxyribonuclease activity at the site of replication (the nucleus).     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

Bridged oligonucleotides as molecular probes for investigation of enzyme-substrate interaction and allele-specific analysis of DNA

The efficiency of enzymatic conversion of DNA complexes containing non-nucleotide inserts has been studied. T4 DNA ligase and Taq DNA polymerase have been included in the study as examples of widely used DNA-dependent enzymes. A series of substrate DNA complexes have been formed using native oligonucleotides and bridged ones bearing non-nucleotide inserts based on phosphodiesters of di-, tetra-, or hexaethylene glycol, 1,5-pentanediol, 1,10-decanediol, and 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran. The perturbation in DNA located far from the site of the enzyme action had almost no influence on the substrate properties of the complex, while insertion near this site significantly deteriorated them. The use of a series of modified duplexes allows one to locate the position of the enzyme-binding site on DNA substrate with the accuracy of 1–2 nucleotides. The presence of a non-nucleotide insert in the complex has been also shown to enhance the efficiency of single mismatch discrimination upon both template-directed ligation and extension of oligonucleotides.     

Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes.

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.     

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).     

Structure of Deoxyribonucleic Acid on the Cell Surface During Uptake by Pneumococcus

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.