Sedimentation In Neutral Sucrose Gradients Research Articles

2-Deoxy-d-glucose Inhibits Rejoining of Radiation-induced DNA Double-strand Breaks in Yeast

Effects of 2-deoxy-D-glucose (2-DG) on radiation-induced DNA double-strand breaks (dsb) have been studied under non-growth conditions in a respiratory-deficient strain of the yeast Saccharomyces cerevisiae. Velocity sedimentation in neutral sucrose gradients was used to measure DNA dsb. Addition of 2-DG to the liquid-holding medium (67 mM phosphate buffer, pH 5, 30 degrees C) at an equimolar concentration with glucose (50 mM) reduced the rate and extent of dsb rejoining. The inhibition of rejoining mediated by 2-DG is reversible for the majority--but not all--of the radiation-induced dsb.

DNA Double-strand Breaks: Their Repair and Relationship to Cell Killing in Yeast

Yeast is a suitable eukaryotic organism in which to study DNA double-strand breakage measured by the neutral sucrose gradient sedimentation technique and cell killing in the same range dose of sparsely ionizing radiations. Radiosensitive mutants (including temperature conditional ones) exist in which rejoining of double-strand breaks (dsb) is not detectable. In such mutants approximately one dsb per cell corresponds to a lethal event, suggesting that a dsb is a potentially lethal lesion. There are two modes by which dsb may confer cell lethality: firstly, an unrepaired dsb may be lethal on its own and secondly, two dsb may interact to form a lethal lesion (binary misrepair). The operationally defined cellular phenomena of potentially lethal damage (PLD) repair and sublethal damage (SLD) repair are both based on the repair of dsb. Induced dsb show a linear and unrejoined dsb a linear-quadratic relationship with dose. At low dose rate the quadratic component is abolished in accordance with the exponential survival curve observed. The dose-rate effect is based on dsb repair during irradiation; it is absent in dsb repair-deficient mutants.

On the Measurement of DNA Double-Strand Breaks by Neutral Elution

DNA double-strand breaks unquestionably increase the rate at which DNA is eluted from cell lysates on a filter, in the neutral elution assay introduced by Bradley and Kohn in 1979 (1). Double-strand breaks are generally agreed to be one of the most biologically important types of DNA damage formed by ionizing radiation, and the neutral elution assay has been widely used to detect such breaks. The purpose here is to bring attention to the possibility that neutral elution may not always give a useful measure of the number of double-strand breaks. This letter may be regarded as a follow-up to a comment by Gunner Ahnstrom in 1985 (2), which has not received sufficient attention. If is it assumed that the rate of DNA elution under nondenaturing conditions is controlled fundamentally by double-strand breaks, then according to several studies, the number varies with dose D of ionizing radiation as aD + fD2 (3-9). The presence of the quadratic term is confirmed in neutral elution measurements on cells irradiated by ionizing radiation, with the assay calibrated with '25I-labeled DNA under the assumption that each 125I decay forms a double-strand break (10, 11). Low-speed neutral sucrose gradient sedimentation, on the other hand, shows a linear relation between radiation-induced double-strand breaks and dose (12-17). Therefore at least one of these methods must be giving wrong answers, and it is appropriate to discuss the underlying theories for the two approaches.

Nuclear protein organization and the repair of radiation damage

A complex network of proteins having attachment sites with DNA are known to exist in mammalian cells and have been referred to as a nuclear cage, matrix, scaffold and nucleoid. Since ionizing radiation is known to induce DNA--protein crosslinks as well as DNA single- and double-stranded breaks, an investigation of the sedimentation of the nucleoid in Chinese hamster ovary (CHO)* cells before, during and after treatments with ionizing radiation was undertaken. Using neutral sucrose gradient sedimentation, it was possible to reproducibly separate the protein and DNA components of interphase nucleoids. Under conditions of radiation damage, the DNA and protein components of the nucleoid were shifted to a coincident position in the gradients consistent with the generation of single- and double-stranded DNA scissions. During DNA damage repair, an apparent recruitment of protein to the nucleoid occurred and a rearrangement of the protein sedimentation was observed as the repair of DNA progressed. These data suggest that the protein component of the nucleoid was dynamic under conditions of DNA damage repair.

DNA double-strand break repair determines the RBE of alpha-particles.

Radiation-induced DNA double-strand breaks (dsb) were studied in Ehrlich ascites tumour cells (EATC) by sedimentation in neutral sucrose gradients at low centrifuge speed. Dsb induction was found to be linear with dose with a frequency of: ndsbmr-1D-1 = (11.7 +/- 2) x 10(-12)Gy-1 for 140 kV X-rays and ndsbmr-1D-1 = (19.1 +/- 4) x 10(-12)Gy-1 for 3.4 MeV 241Am-alpha-particles. Postirradiation incubation of cells under non-growth conditions leads to repair of dsb, reaching a maximum after trep = 24 h. More than 97 per cent of dsb were repaired after an X-ray dose of 25 Gy. The number of residual dsb was found to be a linear-quadratic function of dose: nresmr-1 = (0.0161 +/- 0.0008) x 10(-12)Gy-2D2 for X-rays and nresmr-1 = (1.2 +/- 0.7) x 10(-12)Gy-1D + (0.105 +/- 0.017) x 10(-12)Gy-2D2 for alpha-particles. Thus, after cellular repair the RBE value of alpha-particles was increased from RBE = 1.6 +/- 0.4 (induction of dsb) to a dose-dependent value of RBE = 2.7 +/- 0.4 (at 100 Gy alpha-particles) to 3.8 +/- 1.2 (at 10 Gy alpha-particles) for residual dsb. From the data presented it is concluded that residual dsb are a major cause for loss of the reproductive capacity of EATC after irradiation with X-rays as well as alpha-particles.

Characterization of intracellular DNA strand breaks induced by neocardnostatin inEscherichia colicells

DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.

Evidence for existence and compactness of DNA superstructure units in mammalian cells: A microdosimetric approach to radiation-induced DNA release assayed by neutral sucrose gradient sedimentation

Experimental results obtained with the neutral sucrose gradient sedimentation techniques of analysing mammalian DNA after irradiation in vivo (Chinese hamster cells V79) are evaluated theoretically in order to verify and extend a model by Ormerod and Lehmann that describes the gradual release of free DNA from the DNA-membrane complex. The model is based on the idea of chromatin organization in the form of membrane attached superstructure units (MASSUs) defined by consecutive attachment points in intervals ofM 0 (DNA molecular weight of a MASSU). DNA sedimentation after cell lysis with sarkosyl as detergent allows good separation of the released free DNA from that remaining in the complex. The dose-dependence of both the percentage of DNA released and pertinent molecular weight parameters as measured with X-rays and derived from the model confirms it and yields the MASSU sizeM 0 = 2.4 × 109 g/mol. Furthermore, the model is evaluated with respect to high-LET radiations by a mathematical theory based strictly on microdosimetry. This approach, which could be of general interest as a contribution to theoretical microdosimetry, reduces the measurable quantities explicitly to the single event distribution of lineal energy for the gross volume,V, occupied by a MASSU in the interphase nucleus. Experimental results obtained withα-particles agree with this extended version of the model, yielding 0.2 – 0.3 µm for the diameter ofV. In summary, the following conclusions are drawn: The entire chromatin, at least the greater part of it, must be organized in regular superstructure units with no detectable variance of their lengthsM 0, which is suggested to be a general principle whose functional implications remain to be elucidated.

Frog virus 3 DNA replication occurs in two stages.

Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus.

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa.

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.

Replication of bacteriophage Dp-4 DNA in Streptococcus pneumoniae

The synthesis of viral DNA in diplophage-infected Streptococcus pneumoniae has been studied. A rapidly sedimenting complex was found in detergent lysates of bacteriophage Dp-4-infected cells by sedimentation in neutral sucrose gradients. The complex contained labeled parental viral DNA and newly synthesized DNA. Pulse-chase experiments demonstrated that fast-sedimenting material is metabolically active and serves as precursor for the formation of Dp-4 DNA found free in the cytoplasm and in mature phage particles. This intermediate incorporated most of the radioactivity during a short pulse of [ 3H]thymidine. The complex was dissociated by treatment with Pronase, sodium dodecyl sulfate, and NaOH. Parental DNA did not contribute to mature phage DNA. It is suggested that the fast-sedimenting material represents the replicating phage DNA specifically attached to some other constituent of the infected cell.

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.

DNA replication of bacteriophage θ29: Characterization of the intermediates and location of the termini of replication

Replicative intermediates of the linear duplex of bacteriophage θ29 have been isolated and partially characterized. Replicative DNA, pulse-labeled with [ 3H]thymidine, behaves in neutral sucrose gradient sedimentation and agarose gel electrophoresis as mature θ29 DNA but with a portion of the replicative DNA being of slightly larger size. The faster-sedimenting DNA exhibits an increased buoyant density. In alkaline sucrose gradients, the denatured strands sediment as unit length monomers or smaller fragments. No strands longer than genome length were observed. Sensitivity of the pulse-labeled DNA to S1 nuclease and behavior on benzoylated-naphthoylated DEAE-cellulose (BND-cellulose) chromatography indicate that the intermediates are characterized by extensive single-stranded regions. Intracellular DNA was fractionated on BND-cellulose into completed (double stranded) molecules and replicating (partially single stranded) molecules. Restriction enzyme analysis of the distribution of radioactivity in pulse-labeled completed molecules indicates that termini of replication (most highly labeled regions) are at both ends of the DNA. Electron microscopic analysis of replicating molecules reveals two primary types of intermediates: (1) linear branched molecules with one single-stranded arm and (2) unbranched linear molecules with a single-stranded region extending from one end. These results suggest that θ29 DNA replicates by an asymmetric displacement mechanism. Thus, synthesis is initiated at one end and proceeds in the 5′ to 3′ direction with concomitant displacement of the parental strand of the same polarity. Synthesis of the complementary strand is initiated at the 3′ end of the displaced parental strand. A role for the covalently linked terminal protein in initiation of DNA synthesis is suggested.

Repair of double-strand breaks and lethal damage in DNA of Ustilago maydis.

SUMMARYThe size of nuclear DNA from wild-typeUstilago maydiswas determined to be approximately 6·09 ± 0·3 × 108daltons from neutral sucrose gradient sedimentation analysis. Following exposure to ionizing radiation the nuclear DNA size was reduced due to the production of double-strand breaks in the DNA. These breaks were repaired when the irradiated cells were incubated in medium for at least one hour after irradiation. The repair was seen as a shift in the DNA profile from a low molecular weight region where the control DNA sedimented. Inhibition of protein synthesis by cycloheximide prevented this type of repair. Blocking protein synthesis also decreased the survival of irradiated wild-type cells but not radiation-sensitive mutants. Protein synthesis was necessary within the first one and a half hours after irradiation for the survival of wild-type cells to be unaffected. The results provide additional evidence for an inducible repair process inU. maydis.

Freeze-thaw injury: Evidence for Double strand breaks in [formula omitted] DNA

Neutral sucrose gradient sedimentation studies indicate that freezethawing of Escherichia coli B r results in double strand breakage of its DNA. Breaks were detected after one hour of frozen storage at −16°C, resulting in a distinct displacement of the major peak of [ 3H]DNA toward the meniscus. In addition at this time a minor shallow peak of radioactivity toward the meniscus appeared indicating accumulation of some small molecular weight DNA fragments of heterogeneous size. These small DNA fragments seemed to become the major DNA breakage product after 1 1 2 and 4 months of frozen storage. Double strand DNA breakage continued through 8 and 12 months of frozen storage. However, the sedimentation profile became progressively more broad and shallow suggesting that the initial DNA fragments may have reassociated into molecular aggregates of heterogeneous molecular composition.

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.

In vivo transcription on oligomeric simian virus 40 (SV40) DNA

A simian virus 40 nucleoprotein complex containing an endogenous RNA polymerase activity was extracted from infected BSC-1 nuclei with Triton X-100 in low ionic strength buffer. About 30% of both SV40 DNA and total SV40 transcriptional activity in isolated nuclei are recovered by this procedure. When the nucleoprotein complexes are labeled in vitro with [ 32P] UTP followed by sedimentation in neutral sucrose gradients containing Sarkosyl, labeled RNA is detected as a broad band extending from 21 S to more than 50 S with a major peak at 26 S. After treatment of the labeled complexes with pancreatic ribonuclease, 5 to 8% of the labeled RNA remains associated with the template DNA. The majority of the ribonuclease-resistant material sediments slightly faster than SV40 DNA I. The partially relaxed SV40 DNA I associated with this material represents the predominant template DNA for late SV40 transcription (Birkenmeier et al., 1977). The remainder of the ribonuclease-resistant material sediments in sucrose gradients in a series of faster-sedimenting peaks. The identification of these structures as closed circular oligomeric SV40 DNA molecules that are serving as templates for transcription is based on their sedimentation rates, the time of their accumulation during the lytic cycle, their migration in agarose gels, and their appearance in the electron microscope. At late times after infection at least 10% of the total SV40 template DNA is in the forms of oligomers. The possible correlation between those findings and the presence of high molecular weight virus-specific RNA is discussed.

Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

Parental and replicating vaccinia DNA molecules, labeled with [ 3H]thymidine or [ 14C]thymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90–92 S) were “nicked”; parental DNA molecules sedimenting at 70–72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90–92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2–3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-1 nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes.

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.

Detection of strand breaks in phiX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens.

Supercoiled DNA duplexes of phages phiX 174 and PM2 were treated in aqueous solution at neutral pH with ultimate and proximate carcinogens. Subsequently, the carcinogen-treated phage DNAs were subjected to velocity sedimentation in neutral and alkaline sucrose to quantitative introduction of single strand breaks. Reaction of phage DNA with the ultimate carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr), 7-bromomethyl-benza[a]-anthracene, N-acetoxy-2-acetylaminofluorene [(Ac)2ONFln] and K-region oxides for short periods followed by sedimentation in neutral sucrose gradients led to very few breaks. Incubation with the proximate carcinogens N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene, 7-methyl-, and 7,12-dimethyl-benza[a]anthracene did not result in breaks. However, when the phage DNAs were reacted with the ultimate carcinogens under the same conditions but subsequently alkali-denatured and sedimented in alkaline sucrose gradients, single strand breaks were readily introduced. Incubation with the proximate carcinogens followed by alkali denaturation and sedimentation in alkaline sucrose showed that only 7,12-dimethyl-benz[a]anthracene and, to a minor extent, 7-methyl-benz[]anthracene caused alkali-inducible breaks. The ability of N-methyl-N'-nitro-N-nitrosoguanidine to effect breakdown of superhelical phage DNA in alkali was found enhanced in the presence of N-acetyl-cysteine.