Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.     

Purification and properties of two deoxyribonucleases of Pseudomonas aeruginosa.

A survey of the major deoxyribonucleases in Pseudomonas aeruginosa strain PAO was undertaken. Two activities predominated in Brij-58 lysates of this organism. These have been purified from contaminating nuclease activities, and some of their properties have been elucidated. The first was a nuclease that degraded heat-denatured deoxyribonucleic acid (DNA) to mono- and dinucleotides. The activity of this enzyme was confined to single-stranded DNA, and 100% of the substrate was hydrolyzed to acid-soluble material. The Mg2+ optimum is low (1 to 3mM), and the molecular weight is 6 X 10(4). The second predominant activity was an adenosine 5'-triphosphate (ATP)-dependent deoxyribonuclease. This enzyme had an absolute dependence on the presence of ATP Mg2+ concentrations of approximately 10 mM. Five moles of ATP was consumed for each mole of phosphodiester bonds cleaved. The acid-soluble products of the reaction consisted of short oligonucleotides from one to six bases in length. Only 50% of the double-stranded DNA was rendered acid soluble in a limit digest. The molecular weight of this enzyme is 3 X 10(5). The observation of these enzymes in P. aeruginosa is consistent with the possibility that recombinational pathways similar to those of Escherichia coli are operating in this organism.     

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.     

Study of barley endonucleases and alpha-amylase genes

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.     

Further characterization of a DNA polymerase activity in mouse sperm nuclei.

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.     

Interactions Between Exogenous Deoxyribonucleic Acid and Membrane Vesicles Isolated from Bacillus subtilis 168

Membrane vesicles isolated from competent cultures of Bacillus subtilis 168 bound up to 20 mug of double-stranded deoxyribonucleic acid (DNA) per mg of membrane protein in the presence of ethylenediaminetetraacetate. The formation of the DNA-membrane complex was time, temperature, and pH dependent. Eighty per cent of the DNA could be removed from the complex by treatment with deoxyribonuclease I. Nevertheless, the DNA that remained attached to the vesicles appeared to have been attacked by the enzyme, suggesting that all the complexed DNA is located at the outer surface of the vesicles. Pretreatment of DNA with deoxyribonuclease I destroyed its affinity for the vesicles. The extent of binding decreased by the addition of Mg(2+) ions, especially at high DNA concentrations (more than 2 mug/ml). This effect was partially due to membrane-associated Mg(2+)-dependent endonucleolytic activity, which caused double-strand breaks in addition to single-strand nicks, and to exonuclease activity. The endonucleolytic activity was enhanced by heating the membranes at 80 C. DNA-membrane association was not markedly affected by sulfhydryl reagents, but was largely inhibited by formaldehyde. Endogenous competence-stimulating activity did not alter the DNA-binding capacity of the vesicles.     

DNA helicase IV from HeLa cells.

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.     

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.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

Purification and properties of a novel nucleolar exoribonuclease capable of degrading both single-stranded and double-stranded RNA

A ribonuclease that hydrolyzes either linear duplex or single-stranded RNA in an exonucleolytic manner has been partially purified from Ehrlich ascites tumor cell nucleoli and is free from other ribonucleases. The enzyme will also degrade the RNA complement of an RNA X DNA duplex; however, no nuclease activity is observed on linear duplex or single-stranded DNA. The exonuclease acts on RNA nonprocessively from the 3' end releasing 5'-mononucleotides. The enzyme has a broad pH optimum around pH 8.0, requires Mg2+ or Mn2+ (0.06 mM) for optimum activity, and is sensitive to ethylenediaminetetraacetic acid and N-ethylmaleimide inhibition. Monovalent cations including K+, Na+, and NH4+ are inhibitory. Gel filtration studies of this enzyme gave a Stokes radius of 40 A. Sedimentation velocity measurements in glycerol gradients yield a S20,W of 6.0 S. From these values a native molecular weight of 100 000 was calculated. Copurification of the single- and double-stranded activities, identical reaction requirements, and identical heat-inactivation curves strongly suggest that both activities reside with the same enzyme.     

The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid

1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0.1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.     

Cortisol induction of pulmonary maturation in the rabbit foetus. Its effects on enzymes related to phospholipid biosynthesis and on marker enzymes for subcellular organelles

A deoxyribonuclease was purified approx. 800-fold from crude extracts of the bacterium Alcaligenes faecalis. The enzyme requires ATP and Mn2+; ATP could be replaced by any other ribo- or deoxyribo-nucleoside triphosphate, and Mn2+ could be replaced by Mg2+ in 0.1 M-Tris/HCl, pH 8.0 at 37 degrees C. The enzyme could degrade linear duplex or denaturated DNA, but was inactive with closed-circular duplex DNA from bacteriophase PM-2. In the course of nucleolytic activity, ATP was hydrolysed. We have measured deoxyribonuclease and adenoxine triphosphatase activity in the presence of various salts, and found that the amount of ATP hydrolysis associated with a given amount of deoxyribonuclease activity was decreased in the presence of tetraethylammonium ions. Since these ions decrease the stability of the DNA helix, we conclude that one function of the ATP hydrolysis is to unwind the DNA.     

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

Characterization of the deoxyribonuclease activity of diphtheria toxin

Having discovered that the A domain of diphtheria toxin exhibits intrinsic nuclease activity (Chang, M. P., Baldwin, R. L., Bruce, B., and Wisnieski, B. J. (1989) Science 246, 1165-1168), we proceeded to examine the requirements for optimal enzymic expression. In vitro assays with linear double-stranded DNA demonstrated that optimal activity occurs at pH 7.5 and 37 degrees C. A characterization of the stringent cation-dependence of the reaction revealed increasing activity with increasing Mn2+ up to 30 mM. In contrast, activity levels with Ca2+ or Zn2+ alone peaked at 100 microM and with Mg2+ alone at 1 mM. The Zn2(+)- and Mg2(+)-stimulated activities appear to be dependent on trace amounts of Ca2+. Indeed, inclusion of 2 mM Ca2+ plus 3 mM Mg2+ in the reaction buffer promoted a high level of DNA cleavage even though very little cleavage was seen with either cation alone at 2-3 mM. Addition of 20-200 mM NaCl or KCl caused progressive inhibition. Detection of diphtheria toxin nuclease activity under physiologically relevant conditions suggests that it may be operative in vivo and supports our contention that diphtheria toxin-induced cytolysis is not a simple consequence of protein synthesis inhibition, but rather the final step in a cytolytic pathway linked to chromosomal integrity.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

A special deoxyribonuclease activity accompanying competence-inducing activity

A deoxyribonuclease activity accompanies the competence substance isolated from transformableDiplococcus pneumoniae even in well purified fractions. The deoxyribonuclease seems to exhibit a rather different kind of activity from the one found as a major nuclease in a partially purified competence substance. The products of interaction between the enzyme and double-stranded DNA would indicate that the enzyme might act as an “unwindase” on the double-stranded DNA.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses I. Directing Influence of DNA in the Reaction

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3'-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.