Determination of the rate of renaturation of modified DNA by fluorescence depolarization

Fluorescence depolarization was used to measure the rate of renaturation of T2 DNA, which had been modified by chloroacetaldehyde. Rates were measured on DNA samples with 5–15% of the base pairs modified and were found to agree with rates determined by DNA absorbance kinetics at 260 nm. The renaturation rate of a modified T2 DNA was unchanged in the presence of a ninefold abundance of unlabeled calf thymus DNA.     

Promotion of DNA renaturation by a non-DNA factor in crown call tumor DNA preparation.

A 5400-fold excess of tobacco crown gall tumor DNA increased the renaturation rate of $\text{Agrobacterium tumefaciens}$ DNA, whereas, the same excess of healthy plant DNA had no effect on the rate or kinetics of renaturation. Since deoxyribonuclease treatment of the tumor DNA did not remove its ability to accelerate renaturation, the tumor tissue contains a non-DNA factor that increases the rate of renaturation of $\text{A. tumefaciens}$ DNA.     

Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea.

This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lower the Tm by 2.25 degrees C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.60 degrees C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Acceleration of DNA renaturation rates

The rate of renaturation of DNA may be increased by altering the effective solvent volume available for DNA in solution. Accelerations are demonstrated when neutral and anionic dextran polymers are used to exclude volume in DNA solutions. The logarithm of the DNA renaturation rate constant is shown to be proportional to the concentration of dextran polymer and to be proportional to the intrinsic viscosity of a series of dextran polymers of identical chemical composition. A theory is proposed to account for these results.     

Excluded volume effects on the rate of renaturation of DNA

The rate of renaturation of T2 DNA hits been investigated by using complementary DNA strands of different length. The length of the shorter strand ranged from 0.02 to 1.0 times the length of the longer strand. An excluded volume theory is developed to include this type of reaction as well as the DNA–RNA hybridization reaction. Experimental and theoretical rates of renaturation of DNA are found to be in agreement. For the cases studied, the rate was never greater than twice that observed for short strands of the same length renaturing with themselves. The products of renaturation reactions are also considered.     

Physical studies of chloroacetaldehyde labelled fluorescent DNA

The reaction of chloroacetaldehyde with denatured DNA produces a fluorescent DNA where both the adenine and cytosine bases are modified. The rate of modification of DNA by chloroacetaldehyde was measured using the absorption spectrum shift. The depolarization and quantum yield of native DNA and denatured DNA were investigated as a function of temperature.The melting points and the renaturation rates of a series of derivative DNA's were investigated. The melting point was decreased by 1.3°C for each base modified per 100 base pairs corresponding to a 2.8 Kcal destabilizing free energy per mismatched base pair. The renaturation rate of the derivative DNA is reduced by a factor 2 when the melting temperature is lowered by 13°C.     

Renaturation of denatured, covalently closed circular DNA

The rate of renaturation of denatured, covalently closed, circular DNA (form Id DNA) of the phi X174 replicative form has been investigated as a function of pH, temperature, and ionic strength. The rate at a constant temperature is a sharply peaked function of pH in the range of pH 9 to 12. The position on the pH scale of the maximum rate decreases as the temperature is increased and as the ionic strength is increased. The kinetic course of renaturation is pseudo-first order: it is independent of DNA concentration, but falls off in rate from a first order relationship as the reaction proceeds. The rate of renaturation depends critically on the temperature at which the denaturation is carried out. Form Id, prepared at an alkaline pH at 0 degrees C, renatures from 5 to more than 100 times more rapidly than that similarly prepared at 50 degrees C. Both the heterogeneity in rate and the effect of the temperature of denaturation depend, in part, on the degree of supercoiling of the form I DNA from which the form Id is prepared. However, it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction. The renaturation rate was determined by neutralization of the alkaline reaction and analytical ultracentrifugal analysis of the amounts of forms I and Id. The nature of the proximate renatured species at the temperature and alkaline pH of renaturation was investigated by spectrophotometric titration and analytical ultracentrifugation. It is concluded that the proximate species are the same as the intermediate species defined by an alkaline sedimentation titration of the kind first done by Vinograd et al. ((1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1104-1111). Observations are included on the buoyant density of form Id and on depurination of DNA at alkaline pH values and high temperatures.     

The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation.

The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.     

Thermal stability of homologous and heterologous bacterial DNA duplexes

Thermal stability of homologous and heterologous DNA duplexes renatured according to the renaturation-rate method of De Ley et al. (1970) for 35 min or 17 hr, was estimated from the melting profiles of the duplexes. Comparison of the melting points of native and renatured DNA revealed that in the first 35 min of renaturation highly stable homologous duplexes were mainly formed, whereas up to 7% mismatching occurred in duplexes renatured for 17 hr. Up to 8% more mismatching was found in heterologous DNA duplexes of moderately related coryneform bacteria than in homologous ones after 35 min renaturation. It can be concluded that mismatching in heterologous hybrids of closely related DNAs had been restricted to a few % and of moderately related DNAs to approximately 10% in the initial renaturation phase.     

Physical studies on the binding of cis-dichlorodiamine platinum (II) to DNA and homopolynucleotides.

The amount of cis-dichlorodiamine platinum (II) bound to DNAs of varying (dA + dT) content was assayed by both ultraviolet absorbance spectrophotometry and the use of the radioisotope 1 9 5 Pt. Radioisotope labeling indicates twice as much bound platinum as do optical measurements. The molar ratio of bound platinum r at saturation is approximately half the sum of the nearest-neighbor frequencies of all base-pairs that do not contain thymine. We therefore conclude that platinum does not bind to thymine in DNA. Chromatographic studies with (14C) purine-labeled DNA indicate preferential binding of platinum to guanine, followed by binding to adenine. The luminescence properties of DNA and of homopolynucleotides are strongly affected by bound platinum as a result of a heavy-atom effect. A plot of the fluorescence-to-phosphorescence ratio as a function of r gives a saturation binding curve similar to that obtained using 1 9 5 Pt. Ultraviolet irradiation of DNA treated with the platinum compound results in a 30% increase in the rate of formation of thymine homocyclobutadipyrimidine. When acetophenone sensitization is employed, platinum binding enhances cytosine homocyclobutadipyrimidine formation 10-fold presumably because the triplet level of cytosine complexed with platinum is lowered below that of acetophenone. The viscosity of DNA decreases sharply upon binding platinum, with half the change occuring when less that 6% of the bases are complexed. From the rate of reaction with formaldehyde, we conclude that binding of the platinum compound to DNA induces small denatured regions that unwind in the presence of formaldehyde with a rate about 40 times slower than that of a single-strand chain break.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

DNA conformational kinetics

A model for the time dependence of DNA conformational state probabilities is formulated in the form of first-order differential equations. This model is applied to investigate the renaturation and denaturation rates for T2 and T7 DNA as reported in the series of experiments by Record and Zimm. Qualitative agreement is found in denaturation and for series of renaturation experiments with the same initial condition. However, partial agreement with series of renaturation experiments having the same final condition is obtained only by including an initial bimolecular step with properly matched pairs of strands. Comparison of all experiments with the calculated rates yields 5 × 10⁴ min^?1 as the step rate for melting a single base pair.     

The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.     

Specificity of DNA base release by bleomycin.

DNA was treated with bleomycin in the presence of Fe2+ and 2-mercaptoethanol under conditions where only a few percent of the bases were released. Release of all four bases was a linear function of bleomycin concentration, but the amount of thymine released was twice that of cytosine, 7 times that of adenine, and twelve times that of guanine. Unidentified minor products of thymine, of cytosine and of a purine were also released. Bromouracil did not sensitize DNA to bleomycin-induced breakage, and was released at the same rate as thymine.     

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.     

One hundred-fold acceleration of DNA renaturation rates in solution

Solvents which accelerate DNA renaturation rates have been investigated. Addition of NaCl or LiCl to DNA in 2.4M Et₄NCl initially increases renaturation rates at 45°C and then leads to a loss of second-order behavior. The greatest accelerations are seen with LiCl and dilute DNA. Volume exclusion by dextran sulfate is the most effective method of accelerating DNA renaturation with concentrated DNA. Addition of dextran sulfate beyond 10–12% in 2.4M Et₄NCl fails to increase the acceleration beyond approximately 10-fold. Accelerations of 100-fold may be achieved with 35–40% dextran sulfate in 1M NaCl at 70°C. No other mixed solvent system was found to be more effective, although acceleration may be achieved in solvents containing formamide or other denaturants. The acceleration in 2M NaCl occurs without loss of the normal concentration and temperature dependence of DNA renaturation and is also independent of dextran sulfate concentration if sufficient dextran sulfate is used. Dextran sulfate may be selectively precipitated by use of 1M CsCl.     

Kinetics of renaturation of denatured DNA. I. Spectrophotometric results

The kinetics of renaturation of heat- or formamide-denatured DNA have been studied by following the change of optical density at a constant temperature. Solvents of different ionic strength and various DNA samples have been used. At the lower ionic strengths studied, the reaction follows second-order kinetics, substantiating the hypothesis that strands of native DNA separate upon denaturation and recombine during renaturation. As the ionic strength is increased at a constant temperature, the reaction deviates from simple second-order behavior. This appears to be the result of the inhibition to rewinding caused by short helical segments in the denatured DNA which are more stable at the higher ionic strenth.