Single-strand Specific Nuclease Research Articles

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Discontinuous duplication of both strands of virus 2C DNA.

When Bacillus subtilis infected with virus 2C was labeled with short pulses of tritiated uracil (a specific precursor of the hydroxymethyluracil replacing thymine in 2C chromosome) and the cell lysate was fractionated in alkaline sucrose gradients, most of the radioactivity was found within a lowmolecular-weight peak of DNA (“Okazaki pieces”) of about 2×106 daltons. After longer pulses, segments of large size and full strands were the main labeled products. A shift from low to high-molecular-weight nascent DNA took place slowly during the eclipse phase, and rapidly during the maturation phase. This suggested a lagging of the ligase behind the polymerase at early stages. During the entire replication cycle, pulse-labeled DNA was present within the replication fork in its native form with no involvement of proteins. Furthermore, the amount of lowmolecular-weight pulse-labeled DNA hybridizing with the L strand was higher than that annealing with the H strand. To explain this finding it is suggested that the polymerization or the ligation of small segments is faster for one strand than for the other. Finally, the DNA fraction which is resistant to single-strand specific nuclease was about 10% when alkali-denatured Okazaki pieces were neutralized at 4C and was increased about five-fold upon selfannealing. It is concluded that both strands of 2C DNA duplicate in a discontinuous fashion through the formation of Okazaki pieces, which are held by hydrogen bonds initially and subsequently by phosphodiester linkages. It is proposed also that ligase action is faster for the L strand than for the H strand, and that this phenomenon might be related to both the polarity of chain growth and the asymmetry of transcription of 2C chromosome.

Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus

Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.

S 1 nuclease-specific nicking of mitochondrial DNA containing displacement loops

Conditions are described in which the single strand-specific nuclease S 1 selectively nicks mitochondrial DNA containing displacement loops without nicking supercoiled mitochondrial DNA. Using these conditions, the percentage of molecules containing displacement loops can be easily and accurately determined. This method is superior to the traditional electron microscopic examination for assessing the frequency of displacement loop-containing molecules. In addition, this method permits the determination of the relative specific activities of displacement loop and nondisplacement loop-containing mitochondrial DNA after various radioactive labeling protocols. S 1 nuclease is shown to cleave the displaced strand of the displacement loop, to partially degrade the 7S-initiation strand, but not to cleave the parental template strand complementary to the 7S-initiation strand. The final product is a nicked circular molecule with at least two breaks localized within the displacement loop region in only one of the two parental strands.

Sequence complexity and relative abundance of vaccinia virus mRNA's synthesized in vivo and in vitro.

The sequence complexity and relative abundance of vaccinia virus mRNA's, synthesized in vivo and in vitro, have been measured by DNA-RNA hybridization. Up to 42% of [3H]thymidine-labeled virus DNA can be protected from digestion with nuclease S1, a single-strand specific nuclease, after annealing to excess polyadenylylated mRNA obtained at 7 h after infection. In contrast, only 26% of vaccinia virus DNA is protected when hybridized to polyadenylylated RNA obtained at 2 h after infection in the presence of an inhibitor of DNA synthesis. That the 94 kilobases transcribed early are a subset of the 152 kilobases present late was suggested by hybridization of DNA with a mixture of early and late RNAs. Some control of transcription is lost when virus purified by procedures that include sonic treatment is used for infection since under these conditions similar proportions of DNA are protected by either excess early or late RNA. Excess RNA, synthesized in vitro by enzymes within purified vaccinia virus particles, hybridized to approximately the same fraction of the DNA as did RNA present at late times in vivo. A second type of transcriptional control was demonstrated by kinetic analysis of the hybridization of polyadenylylated RNA to labeled DNA. With virion DNA used as the probe, a single abundance class for early RNA, two classes differing 11-fold in abundance for late RNA, and two classes differing 43-fold in abundance for in vitro RNA were found. To be able to detect high-abundance RNAs of very low sequence complexity, labeled complementary DNA probes to early, late, and in vitro polyadenylylated RNA were used. Evidence that, at late times, RNAs totaling 9 kilobases of sequence complexity are present 40 to 500 times more frequently than the bulk of the virus-specific RNA was obtained. In contrast, the highest abundance class of RNA present at 2 h after infection corresponded to 7 kilobases present in only a 13-fold molar excess over the majority of virus-specific sequences. RNA synthesized in vitro was found to contain a small amount of sequence information, approximately 2 kilobases, which occurred 150 times more frequently than the majority of viral sequences. Studies using hybridization of viral DNA to labeled complementary DNA probes also suggested that 52 to 59% of the polyadenylylated RNA present at 2 h after infection and 82 to 92% of that at 7 h are virus specific.

V and C parts of immunoglobulin kappa-chain genes are separate in myeloma.

Publisher Summary DNA segments coding for the amino terminal and carboxyl terminal half of immunoglobulin chains are separate in an embryo, and there occurs specific rearrangement in these DNA segments during differentiation of lymphocytes. While the simplest model would be that the rearrangement brings a V-gene in contiguity with a C-gene, thereby allowing RNA polymerase to transcribe a whole immunoglobulin gene continuously, it has not been directly shown that this is really the case. This chapter presents evidence that shows that variable and constant parts of immunoglobulin light-chain gene of a mouse myeloma cell are separated by a nontranslated spacer. The chapter presents an experiment in which DNA was hybridized from a plasmacytoma with a purified light chain mRNA under conditions that would not permit renaturation of the gene segments. The conditions needed to be such that would digest all single-stranded nucleic acids with single strand specific nuclease S1 and that would determine the size of the DNA segment protected by the mRNA from digestion with S1 nuclease. The results indicated that the ϰ-mRNA from MOPC 321 myeloma contains stretches of polynucleotides that are transcribed from two DNA segments of about equal length that lie separate in the genome.

Conformational studies on wheat embryo 5S RNA using nuclease S1 as a probe.

Ribosomal 5S 32 P-labelled RNA was isolated from imbibing wheat embryos and digested with nuclease S1, a single-strand specific nuclease. The products of enzymic digestion were separated by polyacrylamide gel electrophoresis and identified by fingerprint analysis of their RNase T1 digestion products. The results indicate that the most sensitive portion of the molecule, and hence, the region containing the least helical structure, is close to the 5'-terminus. Similarly, the most resistant portion of the molecule is close to, but does not include, the 3'-terminus. These findings are discussed in relation to proposed models for the secondary structure of 5S RNA.

Analysis of rat repetitive DNA sequences

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.

Responses of mammalian metaphase chromosomes to endonuclease digestion.

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In 3H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.

The nucleotide sequence of threonine transfer RNA coded by bacteriophage T4

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.

Structure of parental deoxyribonucleic acid of synchronized HeLa cells.

We have investigated the structure of parental DNA as a function of the cell cycle phase of HeLa cells. DNA was isolated from synchronized HeLa cells 0, 5, 8, and 12 h after release from a second exposure to 2 mM thymidine. These DNA preparations were characterized by CS2SO4/AgClO4 buoyant density, sensitivity to a single-strand specific nuclease, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1 nuclease. The cultures were staged according to cell cycle phase by measurements of DNA content per cell by flow microfluorometry. The cell cycle phases were G1/S (0-h culture), S (5-h culture), G2 (8-h culture), and G1 (12-h culture). There are no nuclease-sensitive sites in G2. As the cells enter G1, the number increases, with a maximum being reached in the S phase. The number of breaks in DNA with respect to cell cycle phase follows the same pattern. The amount of single strandedness, measured by buoyant density and nuclease sensitivity, is also minimal in G2, increases in G1, with a maximum achieved in the S phase. It appears that there is a chromosomal cycle, reflected as continuous structural changes in the DNA molecule, as cells traverse the cell cycle.

Detection of carcinogen-DNA adducts by radiommunoassay.

COVALENT binding of carcinogen to nucleic acids is believed to be an essential component of the carcinogenic process1, so it is desirable to have highly sensitive and specific methods for detecting such adducts in cells and tissues exposed to known and suspected carcinogens. We describe here a radioimmunoassay (RIA) capable of detecting nanogram amounts of DNA adducts resulting from the covalent binding of the carcinogen N-2-acetylaminofluorene (AAF). AAF and its activated derivative N-acetoxy-AAF (N-Ac-AAF) are potent carcinogens2 and mutagens3,4, and transform cells in culture5,6. DNA obtained from rat liver following in vivo exposure to AAF, and DNA exposed in vitro to N-Ac-AAF contain as the major (80%) adduct N-(deoxyguanosin-8-yl)-acetylaminofluorene (dG-8-AAF)7–9 and a minor (20%) adduct 3-(deoxyguanosin-N2−yl)-acetylaminofluorene (dG-N2-AAF)10. These two types of modification produce markedly different conformational effects on the DNA helix2,11,12. The major adduct, recognised by single strand-specific nucleases in vitro13,14 and DNA repair enzymes in vivo9,15 was used as the immunogen in this study.

In vitro synthesis of a DNA probe for antisense globin sequences.

A DNA probe specific for sequences complementary to globin RNA has been prepared in vitro from globin complementary DNA (cDNA). Globin cDNA was used as the template for the synthesis of a complementary strand (ccDNA) by avian myeloblastosis virus DNA polymerase. After treatment with single strand-specific S1 nuclease, double-stranded globin DNA was denatured in the presence of a vast excess of globin RNA. After hybridization of the cDNA with globin RNA, the ccDNA was isolated by hydroxyapatite chromatography. The ccDNA probe hybridized efficiently to globin cDNA, but not at all to globin RNA. This probe should prove useful in assessing the asymmetry of gene expression in cell-free transcriptional systems.

Conformation of DNA modified with a dihydrodiol epoxide derivative of benzo[a]pyrene.

The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.

Action of single-strand specific nucleases on model DNA heteroduplexes of defined size and sequence

The sensitivity of the model DNAs containing dA-dG and dtg-dG heteroduplex regions of defined length to S1 and mung bean single-strand specific nucleases was tested by polyacrylamide gel electrophoretic analysis of the distribution of product oligonucleotides. Single-base mismatch heteroduplexes were extremely resistant to these nucleases, although low levels of cleavage at the heteroduplex nucleotide were observed at high nuclease concentrations. The nuclease sensitivity of dA-dtg heteroduplex regions increased gradually as the length of the heteroduplex region increased frome one to six nucleotides. The sensitivity of dG-dG heteroduplexes three to five nucleotides long was considerably greater than that of the single dtg-dG mismatch.

Deoxyribonucleic acid structure in human diploid fibroblasts stimulated to proliferate.

When contact-inhibited human diploid fibroblasts that have been maintained on medium containing 0.5% serum are stimulated by 10% serum they leave the G0 state and enter the cell cycle at G1. The onset of DNA synthesis occurs at 12 to 13 h. DNA was isolated from cultures that had been stimulated for 0, 2, 4, 6, 12, 16, 20, 24, and 30 h. These DNA preparations were characterized by sensitivity to a single strand specific nuclease, CsSO4/AgClO4 buoyant density, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1-nuclease. The DNA from 4-h stimulated cultures has an increased amount of single strandedness as indicated by sensitivity to S1-nuclease and buoyant density. The amount of single strandedness increases as the cells enter S-phase and decreases as they enter the G2 period. The DNA isolated from G0 cells behaves as native, duplex DNA with virtually no breaks. The DNA from 2-h and 4-h stimulated cultures contained a significant number of breaks. The number of breaks increases to a maximum in S-phase and declines as the cells enter the G2 period. There are no nuclease-sensitive sites (gaps) in G0 or 2-h cultures. The number of sites increases 4 h after stimulation, reaches a maximum during S-phase, and decreases as the cells enter G2. These results lead us to suggest a chromosomal cycle at the level of DNA itself, whereby structural changes are occurring throughout the entire G1 period, aimed toward the initiation of DNA synthesis. These changes reach a maximum degree during replication and decline as replication is terminated.

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.

Distribution along DNA of the bound carcinogen N-acetoxy- N-2-acetylaminofluorene in chromatin modified in vitro

Chromatin from duck erythrocytes was modified in vitro by the carcinogen N-acetoxy- N-2-acetylaminofluorene (N-Ac-O-AAF). The distribution of the carcinogen along the DNA molecule was studied using staphylococcal nuclease which allows the fractionation of chromatin DNA into two zones. It was shown that the carcinogen binds preferentially to the regions of chromatin sensitive to the enzyme; however, the regions of DNA tightly bound to histones and resistant to the enzyme react comparatively well. The single-strand specific nuclease S 1 which digests DNA modified by the carcinogen in vitro did not digest chromatin under the conditions used. Some possible mechanisms for the interaction of the carcinogen with chromatin are discussed.

Effect of estrogen on gene expression in the chick oviduct. Studies on the initiation of RNA synthesis on chromatin in vitro.

Studies on the effect of the temperature requirement for RNA chain initiation by Escherichia coli RNA polymerase on DNA, chromatin, and reconstituted chromatin were carried out in order to better understand the nature of the initiation process. Varying the temperature or ionic strength during preincubations had little effect on the formation of stable preinitiation complexes (RS complexes) between RNA polymerase and chromatin. This observation was in marked ocntrast to similar studies performed on native DNA and indicates that initiation sites for RNA synthesis on chromatin are different from those on double-stranded DNA and resemble more closely initiation of RNA synthesis on single-stranded DNA. These results suggest that the local unwinding of the initiation region which is required for RNA chain initiation on native DNA may not be a prerequisite for RNA initiation on chromatin. The results of studies on reconstituted chromatin devoid of different classes of chromatin proteins demonstrate that both histone and nonhistone fractions are essential in maintaining the characteristics inherent to initiation of RNA synthesis on chromatin. Removal of either moderatley lysine-rich histone or arginine-rich histone fractions led to the complete loss of the characteristic "chromatin type" initiation pattern for RNA synthesis, whereas, removing lysine-rich (Fi) histone had no effect. Single strand specific and double strand specific antibodies to DNA and a single strand specific nuclease were used as probes to examine the structure of chromatin sites for initiation of RNA synthesis. Their effects on RNA synthesis suggest that initiation sites on chromatin are not located in freely accessible single-stranded or double-stranded regions of DNA.