Unwinding Rate Research Articles

DNA Conformation of Chinese Hamster V79 Cells and Sensitivity to Ionizing Radiation

Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.

High glucose induces DNA damage in cultured human endothelial cells.

Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells--but not from fibroblasts--exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) cells vs. 88 +/- 42 in control cells, mean +/- SD, P = 0.04), which is indicative of increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage--a process that is under genetic control--may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication.

DNA strand breaks induced by low-energy heavy ions.

The DNA unwinding method was used to estimate DNA breakage in Chinese hamster cells exposed to heavy ions with LET in the range of 750-5000 keV/micron. Comparison of the primary induced unwinding rate per dose unit for ions with various track diameters but similar LET showed a pronounced influence on the track diameter. Low-energy ions, producing thin tracks with diameters (penumbra) in the submicrometer region, were almost two orders of magnitude less efficient than more energetic ions producing tracks with diameters of several micrometers and about three orders of magnitude less efficient than X-rays. For the thin tracks, clustering of breaks was indicated by comparison of the DNA unwinding rates in two different alkaline solutions. The results indicate that the unwinding rate cannot be used as a good measurement for DNA breaks in this case. The residual unwinding remaining after 4 h of repair at 37 degrees C correlated well with the ability of the various ions to produce cell-killing.

Rapid analysis of DNA strand breaks in soft tissues

A technique was developed to measure rapidly DNA strand breaks in soft tissues. This method measured the rate of alkaline unwinding of DNA, which was proportional to strand breakage. Alkaline unwinding of DNA was done by treating tissue homogenates with NaOH. Single-stranded DNA was removed by extraction with aqueous phenol. DNA unwinding was quantitated by measuring the remaining double-stranded DNA. Using the described technique, a dose-effect relationship was observed between N-nitrosodimethylamine (NDMA) and alkaline unwinding of mouse liver DNA.

Effect of Intercellular Contact on DNA Conformation, Radiation-Induced DNA Damage, and Mutation in Chinese Hamster V79 Cells

Chinese hamster V79 cells, when grown as small spheroids in suspension culture, are more resistant to killing by ionizing radiation than when grown as monolayers. We have attempted to determine whether this enhanced survival following irradiation is reflected in DNA damage and repair at the structural level (by measuring alkali-induced DNA unwinding rates from strand breaks) and at the functional level (by measuring resistance to forward mutation at the HGPRT locus). For a given dose of radiation, the unwinding of DNA in high salt/weak alkali was less complete for spheroid DNA than for monolayer DNA, and the rate of repair of radiation damage was faster in spheroid DNA. These differential responses were lost 8 hr after separation of spheroids into single cells, coinciding with loss of radioresistance measured by clonogenicity. In addition, spheroid cells showed fewer numbers of induced mutants per Gray, although, for a given level of survival, the mutation frequency for monolayers and spheroids was identical. These results suggest that conformational changes in DNA resulting from cell growth as spheroids might enhance repair of radiation-induced lesions.

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes.

Activation of quiescent human peripheral blood lymphocytes or purified T cells by the mitogen, phytohemagglutinin (PHA), involves a rapid rejoining of DNA breaks present in the resting cells as detected by both nucleoid sedimentation analysis and rate of strand unwinding in alkali. Inhibitors of the enzyme ADP-ribosyltransferase (ADPRT) prevent activation of peripheral lymphocytes or T cells by PHA or concanavalin A in a dose-dependent manner, but only if present during the early stages. They do not affect subsequent proliferation if added later, nor do they inhibit the growth of lymphoblastoid cell lines. The inhibitors slow the rejoining of DNA breaks but do not affect the binding of mitogen to the cell surface or the early PHA-stimulated turnover of plasma membrane inositol phospholipids. DNA breaking and rejoining, regulated by ADPRT, may be involved in controlling gene expression during differentiation.

Unwinding of DNA induced by fluorescein mercuric acetate.

AbstractFluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double‐strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10−5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single‐strand breaks and a smaller number of double‐strand breaks, also increased the initial rate. However, this increase was due only to the double‐strand breaks, that is, single‐strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 105 erg/mm2, suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.

DNA damage in ultraviolet-irradiated HeLa and CHO-Kl cells examined by alkaline lysis and hydroxyapatite chromatography

DNA damage and repair activity have been examined in cultured mammalian cells by the technique of alkaline lysis followed by analysis of the single-strand content of DNA by hydroxyapatite chromatography; the rate of unwinding of DNA in alkali is related to the number of single-strand breaks present. When HeLa cells are ultraviolet-irradiated and incubated, on subsequent alkaline lysis the DNA unwinds at a faster rate than the DNA of unirradiated cells. The rate is increased if inhibitors of DNA synthesis (hydroxyurea, deoxyadenosine or 1-β- d-arabinofuranosyl cytosine) are present during the incubation. Similar effects are seen in CHO-Kl cells synchronised in G1 phase, though mitotic CHO-Kl cells show little effect of post-irradiation incubation or of the presence of inhibitors. It appears that agents known to block replicative DNA synthesis can also inhibit repair DNA synthesis following ultraviolet irradiation, leading to an accumulation of single-strand breaks in DNA produced by repair nuclease activity. This phenomenon is probably responsible for the retarded sedimentation of such DNA on alkaline sucrose gradients.

Rupture of base pairing in double-stranded poly(riboadenylic acid)·poly(ribouridylic acid) by formaldehyde: medium chain lengths

By assuming that the opening of hydrogen bonds due to thermal fluctuations is a very fast step and that the reaction of formaldehyde with the imino or amino group is a slow step, we have constructed a model for the unwinding process of poly(A-U) induced by formaldehyde. The denaturation equation derived from the model is essentially the same as that of the zipper model for moderately long chain lengths. The model predicts the following phenomena which are in agreement with our experimental findings. The rate of unwinding is approximately first order for unfractionated polynucleotides and zero order for fractionated samples. This means that formaldehyde ruptures helical residues sequentially starting from the ends and working toward the center. Our model further predicts that the denaturation rate is linearly dependent on -log[Na+] and pH at low ionic strength and is almost independent of [Na+] and pH at high ionic strength. Spectrophotometric measurements on poly(A-U) were done to confirm our theoretical findings.

Rate of unwinding small DNA

The kinetics of the helix-coil transition of low molecular weight DNA was investigated using temperature-jump techniques. The material was sonicated or sheared, then fractionated according to molecular weight using differential solubility in an isopropanol-buffer mixture. To prevent irreversible strand separation the samples were cross-linked with mitomycin C. Kinetic observations (266 nm) were made as a function of molecular weight, fraction coil, perturbation size and salt concentration. The Eigen-DeMaeyer apparatus was used for times between 5 μsec and 2 sec; slower effects required a special instrument. Decay times varying with M 1.9 down to 6.0 × 10 5 daltons are attributed to fractionally limited unwinding. Some faster effects are from overtones (melting from ends). The rate of unwinding of DNA with a molecular Weight of 1.0 × 10 5 daltons appears to be limited by the breakage of hydrogen bonds. From the data we estimate a rate constant of 4 × 10 6 sec −1 for base pair formation. A very fast decay (~30 μsec) is possibly the nucleation of coil at an A + T rich region. Ascribed to protolytic equilibria and to non-specific base-base interaction is an irresolvable (<5 μsec) optical density change. Also of interest is the appreciable contribution of very slow effects (some slower than 10 2 sec). These are nearly independent of molecular weight. We believe this originates from the difference in modes of melting for G + C rich and A + T rich regions of amolecule; the former approaches an “all-or-none” transition. The A + T rich regions, because they are less stable, melt first, leaving G + C rich regions between loops. The merging of two of these loopsis statistically favorable but this requires the breakage of a large number of base pairs. Thus, this melting is entropically driven against a large activation energy barrier giving rise to exceedingly slow kinetics.

The rate of DNA unwinding

We report three kinetic effects in the response of large DNA molecules to temperature perturbations ranging in size from 0.25 to 18 deg. C. Experiments were done in the alkaline melting transition, and the rate was followed by observing the change in absorbance. The temperature increase was produced by passage of an electric current through the solution, using the standard temperature-jump technique for times up to one second. For the time range from 1 second to hours a special instrument was constructed, with which the temperature in the cell can be increased by up to 20 deg. C in less than one second, and maintained indefinitely. The fastest kinetic response of the DNA, called “instantaneous”, occurs in less than 20 msec, and increases in amplitude as the size of the temperature jump increases. We associate this process with a rapid structural disorganization which does not require unwinding of the molecule, analogous to melting of a closed double circle of DNA. When the perturbation size is moderate (between 6 and 18 deg. C) the initial instantaneous effect is followed by kinetic curves of homogeneous appearance, which we label the “fast” effect. The characteristic time for this process (determined as a linear average of the component exponential decay times) depends on the 2.3 power of the molecular length. We ascribe rate-determination for this effect to viscous resistance to unwinding the double helix. Because of the strong dependence on molecular weight, kinetic measurements under these conditions can be used to detect small amounts of undegraded DNA in the presence of material that is broken or contains single-strand breaks. Small perturbations within the melting transition show a kinetic component which is nearly independent of molecular weight and, for a DNA like that from T7 bacteriophage, much slower than the fast effect. This process, labeled the “slow” effect, has an apparent activation energy of at least 100 kcal., and is therefore not likely to be primarily friction-limited. We ascribe it to one of the nucleation events in the co-operative melting transition, specifically the disappearance (or de-nucleation) of a helical section separating two coil regions.

KINETIC STUDIES ON THE THERMAL DENATURATION OF DEOXYRIBONUCLEIC ACID WITH A HIGH-FREQUENCY HEATING METHOD.

The rate of heat denaturation of calf-thymus, salmon-sperm and bacterial DNA's was studied with a high-frequency electric heating system. It was found that the rate of heat denaturation of DNA is rather slow, requiring more than 3 sec. The rate of denaturation is dependent on the guanine-cytosine content, and it is faster when the guanine-cytosine content is smaller. The rates are also faster below pH 5.0. Furthermore, the rate is faster in the presence of 8 M urea in the case of salmon-sperm DNA but is not affected by urea in the case of calf-thymus DNA. The relationship between the rate of unwinding and the length of DNA is also investigated but because of the possible presence of single-strand breaks, the results are complicated and no conclusion is reached concerning this problem.

Polymerization kinetics of biological macromolecules on templates.

A kinetics of formation of polymers on linear templates is developed which is applicable to the synthesis of biological macromolecules. Two extreme situations are given theoretical treatment. In both, monomer diffuses to the template surface where polymerization may occur if an adjacent site is suitable occupied. In addition the possibility of monomer desorption is included. In one model (``unrestricted'') the reaction is initiated on each template by the polymerization of two monomers and proceeds through the addition of monomer to growing ends or by the coupling of the growing chains. In the second (``restricted'') model the number of growing centers per template is zero or one and growth is achieved through monomer addition solely. In both cases the template is assumed to be of uniform size and either single stranded or, if double stranded, with an unwinding rate that is rapid in comparison with the polymerization processes. The number of template molecules is assumed constant during the synthesis, which disregards the function of new chains as sites for continued synthesis. All degradation mechanisms are omitted from consideration, which may restrict the present theories to low conversion processes. The pronounced differences between the unrestricted and restricted formulations in respect to kinetics and product molecular weight are elaborated and discussed in relation to recent experimental results on DNA synthesis in vitro. Finally, from known replication times in vivo, estimates of the rate parameters in the two models are derived.

Rate of unwinding of DNA

The mean time of unwinding of a DNA molecule is calculated. The unwinding is assumed to begin at one end and to proceed under the combined forces of diffusion and a driving torque of arbitrary magnitude. The standard deviation of the unwinding time is calculated approximately. If the free energy decrease on unwinding one turn, in units of kT , times the number of turns in the molecule is a number much greater than unity, diffusion plays a negligible role in the unwinding.

The formation of hybrid DNA molecules and their use in studies of DNA homologies

Heavy-isotope-labeled DNA has been used to study strand separation and recombination. The rate at which 14 N− 15 N biologically half-labeled DNA can be made to separate into subunits corresponds very closely to what has been predicted for the rate of unwinding of the strands of a double helix. The use of a phosphodiesterase from E. coli ( Lehman, 1960 ) which selectively attacks single-stranded DNA has made it possible to remove unmatched single chain ends from renatured DNA and reduce the remaining differences between renatured and native DNA. A mixture of heavy-isotope-labeled and normal bacterial DNA was taken through a heating and annealing cycle, treated with the phosphodiesterase and examined by cesium chloride density-gradient centrifugation. Three bands were observed, corresponding to heavy renatured, hybrid, and light renatured DNA. As would be expected for random pairing of complementary strands, the amount of the hybrid was double that of either the heavy or the light component. It has thus been demonstrated that the strands which unite in renaturation are not the same strands that were united in the native DNA but instead are complementary strands originating in different bacterial cells. The formation of hybrids has been possible only where the heavy and normal DNA samples have a similar overall base composition. It has also been shown for DNA samples isolated from bacteria of different genera in a case where genetic exchange by conjugation has been demonstrated. The evidence for the parallelism between genetic compatibility and the formation of DNA hybrids in vitro has led to the proposal that organisms yielding DNA which forms hybrid molecules are genetically and taxonomically related.