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Results for “DNA Breaks, Double-Stranded”

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PubMed1978

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength.

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.

Animals↗

PubMed1976

Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency.

A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.

DNA Repair↗

PubMed1977

Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells.

A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.

Adenosine Triphosphate↗

PubMed1975

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.

Computers↗

PubMed1978

Gene 4 protein of bacteriophage T7. Characterization of the product synthesized by the T7 DNA polymerase and gene 4 protein in the absence of ribonucleoside 5'-triphosphates.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze extensive DNA synthesis on duplex phage T7 or PM2 DNA templates containing single strand breaks. A variety of physicochemical techniques have been used to characterize the DNA product synthesized in this reaction in the absence of ribonucleoside 5'-triphosphates. Pyknographic and sedimentation analyses reveal that all of the newly synthesized DNA is covalently attached to the template DNA. Analysis by electron microscopy shows the major portion of the product molecules synthesized on duplex T7 DNA templates to consist of a double-stranded branch attached to an intact template molecule. Using PM2 DNA templates, the predominant product consists of a double-stranded branch attached to the circular PM2 DNA template. Analyses of these product molecules indicate that DNA synthesis by the gene 4 protein and T7 DNA polymerase is initiated at single strand breaks in the duplex DNA and that synthesis is accompanied by extensive displacement of one of the parental strands. At later times in the reaction, a portion of the 3'-hydroxyl terminus of the newly synthesized DNA is displaced from the template by branch migration and is used as a primer by the DNA polymerase to copy the displaced 5' single-stranded parental strand to form a duplex branch.

Coliphages↗

PubMed1979

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.

Bacteriophages↗

PubMed2026

Chromosome-scale genome remodeling in tumor evolution: Copy number alterations and structural variants as two sides of the same coin.

Chromosome-scale genomic rearrangements are a dominant force in tumor evolution. Copy-number alterations (CNAs) and structural variants (SVs) constitute two complementary axes of this process. Although detection technologies now deliver near-comprehensive catalogs, technical resolution has outpaced conceptual integration. In this review, we frame CNAs and SVs as inextricable facets of chromosomal aberrations. They reshape cancer genomes through altered gene dosage and three-dimensional regulatory rewiring. CNAs quantify the gene-dosage imbalance, yet arise through mechanistically distinct routes. Segmental CNAs typically require chromosomal breakage, and therefore often coincide with SV junctions. By contrast, whole-chromosome aneuploidy and whole-genome doubling (WGD) primarily reflect mitotic or cytokinetic failure and can occur without local breakpoints, while nevertheless reshaping the karyotypic landscape and seeding subsequent structural complexity. SVs, in turn, range from unbalanced events that alter copy number to ostensibly balanced exchanges that predominantly rewire regulatory architecture. Despite their diverse and sometimes catastrophic architectures, SVs are ultimately rooted in double-strand break formation and error-prone resolution. By integrating CNAs and SVs within a unified mechanistic and functional framework, we aim to convert catalogs into concepts and distill the organizing principles that govern tumor genome evolution.

Humans↗

PubMed2025

Enhanced cleavage of genomic CCR5 using CASX2Max.

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2Max in cellular gene excision. In sum, CasX2Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

Humans↗

PubMed1976

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.

Centrifugation, Density Gradient↗

PubMed1978

Degradation of nucleic acid in aqueous solution by ionizing radiation. III. The correlation of radiation damage with change in melting transition--model experiments.

The melting behavior of polydeoxynucleotide double helices of known structure is analyzed in terms of the thermodynamics of helix stability, taking into account separately those contributions to the transition free energy that are proportional to the numbers of polymer molecules and those that are proportional to the numbers of base pairs formed. From the analysis of the melting transitions of helices having an alternating (d-)A.T, G.C base-pair sequence and containing either single-strand nicks or both nicks and damaged thymine bases, the effects of these structural lesions are assessed; it is concluded that, in a moderately long helix of this sequence (400 base pairs), the initial introduction of one mid-chain double-strand break or single-strand break produces respectively some 3.5 or 4 times as much depression in the transition temperature (Tm) as does the destruction of a single internal A.T base pair.

DNA, Single-Stranded↗

PubMed1977

Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences.

We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.

Animals↗

PubMed1978

Studies on the single-stranded discontinuities of the cauliflower mosaic virus genome.

The Cauliflower Mosaic Virus (CaMV) genome is a double-stranded DNA molecule of about 5 million daltons. Native DNA molecules appear heterogeneous when analysed by gel electrophoresis. We have examined the nature of this apparent heterogeneity. Besides, this genome is shown here to contain three single-stranded breaks, as revealed by different denaturation experiments: heating at 75 degrees C, treatment with NaOH or dimethyl sulfoxide (DMSO). Labelling with terminal transferase proves that the 3' ends at these interruptions all have free hydroxyl groups. Electron microscopy and alkaline gel electrophoresis indicate that these three discontinuities are shared by both strands, and that they are not randomly located. S1 nuclease is active on CaMV DNA and generates three fragments. The comparison between the sizes of these fragments and of the products of denaturation leads us to consider that S1 acts at the level of the interruptions. We have determined that two of them, distant by one third genome unit, are in the same strand; the other is in the opposite strand, distant by one sixth genome unit from the nearest other one. The combined use of restriction enzymes and S1 nuclease has enabled us to locate these three discontinuities on the restriction map of the CaMV genome that we have otherwise established.

Chromosome Mapping↗

PubMed1979

The response of ataxia telangiectasia cells to bleomycin.

The autosomal recessive disorder, ataxia telangiectasia (AT) is characterised by cellular sensitivity to ionizing radiation. The molecular basis of this radiosensitivity is the subject of controversy. We report here that cultured fibroblasts from AT patients are also sensitive to the lethal effects of bleomycin. As with ionizing radiation, no defect has been observed in the overall rejoining of single or double-strand breaks produced by bleomycin. Since, however, only apyrimidinic (and to a lesser extent apurinic) sites and strand breaks are known to be produced by bleomycin, we tentatively suggest that AT cells are unable to rejoin a very small fraction of the total strand breaks. We attribute our inability to detect such unrejoined strand breaks to the relative insensitivity of the sucrose gradient procedures normally used to detect strand breaks.

Ataxia Telangiectasia↗

PubMed1979

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.

Adenoviruses, Human↗

PubMed1979

Purification and characterization of an endonuclease specific for single-stranded DNA from Bacillus subtilis Marburg.

Bacillus subtilis Marburg TI (thy,trpC2) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease MII), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000:1 by comparing the initial rates of introducing single-strand breaks into the DNA's.

Adenosine Triphosphate↗

PubMed1979

Deoxyribonucleic acid strand breaks during drying of Escherichia coli on a hydorohobic filter membrane.

Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures. A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above. Sucrose gradient studies showed that single- and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above. These results were observed in all cells rehydrated with 0.03 M tris (hydroxymethyl) aminomethane-hydrocholoride buffer solution at 0 or 37 degrees C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution. Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry. Removal of unfreezable water of cells in the drying, therfore, might induce deoxyribonucleic acid strand breaks.

DNA Repair↗

PubMed1977

Evaluation of a standardised assay for the measurement of antibodies to double-stranded (native) DNA.

A standardised commercially available radioimmunoassay kit for the detection of antibodies to native DNA (N-DNA) has been evaluated in clinical practice. This test system is shown to be a reliably reproducible method of detecting these antibodies. In addition, evaluation of the purity of the radiolabelled test antigen in this assay has shown it to be almost entirely double stranded (native) DNA with virtually no contamination with single stranded (denatured) DNA, and with few areas of single stranded breaks or ends in the duplex molecule. The inclusion of known standards and precise characterisation of the DNA has partially overcome variability in results and provides for interlaboratory standardisation which is lacking in the techniques used at present.

Antibodies, Antinuclear↗

PubMed1976

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells.

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.

Cell Line↗