The properties of native and denatured DNA in buoyant rubidium trichloroacetate at neutral pH

Aqueous RbTCA is generally suitable as a buoyant solvent for both native and denatured DNA at neutral pH and room temperature. Native PM-2 DNA II, for example, is buoyant at 3.29 M salt, 25 degrees C; whereas the denatured strands band together at 4.52 M. Two properties of the solvent make this system uniquely useful for separations based upon the extent of secondary structure. First, the melting transition temperature for chemically unaltered DNA is depressed to room temperature or below. Second, the buoyant density increase accompanying denaturation is extraordinarily large, 174 mg/ml for PM-2 DNA II. This value is three times that found in aqueous NaI and ten times that for CsCl. The properties of the RbTCA buoyant solvent presented here include the compositional and buoyant density gradients and the buoyant density dependence upon base composition. The DNA remains chemically unaltered after exposure to RbTCA as shown by the absence of strand scissions for closed circular DNA and by the unimpaired biological activity in transformation assays. Intact virion DNA may be isolated by direct banding of whole virions in RbTCA gradients without prior phenol extraction. Strongly complexed or covalently bound proteins may be detected by their association with the buoyant polymer in the denaturing density gradient.     

The helix-coil transition of closed and nicked DNAs in aqueous neutral trichloroacetate solutions.

The melting transition for closed, underwound DNAs and for nicked or linear DNAs was monitored by velocity sedimentation and by absorbance spectroscopy in aqueous NaCCl3CO2 (NaTCA) and RbTCA. The addition of neutral trichloroacetate lowers the midpoint of the helix-coil transition by 26% C/M for RbTCA and by 32% C/M for NaTCA, depressing the denaturation region to near room temperature at neutral pH. The melting of nicked DNA is cooperative, occurring over a temperature range of about 5.6 degrees C. The melting profile for closed DNA is broad and noncooperative with a transition breadth greater than 45 degrees. Closed DNAs undergo a structural alteration, as revealed by velocity sedimentation, resulting in a reduction in the number of superhelical turns at temperatures and salt concentrations substantially below the melting temperatures and salt concentrations substantially below the melting temperature of the nicked DNA. The reduction in the extent of supercoiling continues upon isothermal addition of salt up to the salt concentration at which all superhelical turns are removed. The salt concentration at the principal minimum in the sedimentation velocity profile (3.16 M NaTCA for PM-2 DNA) is approximately the same as that at the midpoint of the helix-coil transition for the nicked DNA.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans.

The effects of temperature and ethidium bromide on the banding of heat-denatured DNA was studied during equilibrium centrifugation in density gradients of NaI. Centrifugation at 10 degrees C prevents the partial renaturation of Escherichia coli DNA and Clostridium perfringens DNA that occurs at 20 degrees C. A centrifugation temperature of --5 degrees C is required to prevent renaturation of T7 phage DNA. Ethidium bromide decreases renaturation of Escherichia coli DNA during centrifugation at 20 degrees C and causes a small shift in the buoyant density of both denatured and native DNA. Equilibrium centrifugation at lower temperatures prevents DNA renaturation and permits increased utilization of the large buoyant density difference between native and heat-denatured DNA in gradients of NaI.     

Measurement of superhelix densities in buoyant dye/CsCl. The use of a standard other than native SV40 DNA.

The dye-induced separation between closed and open duplex DNAs in buoyant CsCl is determined primarily by the superhelix density of the closed DNA, provided that all other experimental variables (such as the solution density and dye concentration) are held constant. The extent of the buoyant separation may be used to estimate the superhelix density of an uncharacterized closed DNA, by comparison with the corresponding separation with native SV40 DNAs under identical conditions. We present here an extension of these quantitative relationships to permit the use of an arbitrarily selected closed duplex DNA of known superhelix density, with the accompanying open form, as a reference. The general result is that the ratio of buoyant separations for any two closed/open DNA pairs remains a linear function of the difference in superhelix densities between the closed DNAs. The value of the proportionality constant depends, however, upon the magnitude of the superhelix density of the closed DNA selected as reference.     

An endonucleolytic activity of nuclease TT1 specific for superhelical DNA.

Highly purified nuclease TT1 from T. thermophilus HB8 acts on a linear single- and double-stranded DNA as an exonuclease and produces 5'-mononucleotides either from the 5'- or 3'-terminus. It was found that the enzyme also possesses an endonuclease activity specific for superhelical (form I) and single-stranded circular DNA. Form I of various kinds of DNA (phi X174, PM2, Co1E1 and RF 1010 etc.) is nicked to yield first relaxed circles (form II) and then nicked at the opposite site to yield unit length linear DNA (form III), which is subsequently hydrolyzed from the 5'- or 3'-terminus. A single cleavage of the form I of phi X174 DNA seemed to occur at a limited number of unique sites. Both endonuclease and the known exonuclease activities co-migrate on polyacrylmide gels, show the same pH and temperature optima, are stimulated by Mg2+ and are inactivated by EDTA similarly.     

Temperature dependence of the gel electrophoretic mobility of superhelical DNA.

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a function of both temperature and of linking number (Lk). At temperatures below 37 degrees, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37 degrees the electrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37 degrees - 65 degrees the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37 degrees and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6 degrees, which lies within 3 degrees of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.     

A new method for the purification and identification of covalently closed circular DNA molcules.

A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.     

High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8 degrees, compared to 7.2 degrees for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (sigma = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6 degrees. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DNA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6 degrees. Moderately negatively supercoiled DNA (sigma = -0.018), but not moderately positively supercoiled DNA (sigma = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate.

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.     

Identification of a potent decatenating enzyme from Escherichia coli

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.     

Phosphodiesterase I in human urine: purification and characterization of the enzyme

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

The replication of kinetoplast DNA networks in Crithidia fasciculata

Kinetoplast DNA from the mitochondria of Crithidia is in the form of a two-dimensional network of thousands of minicircles each containing about 2.5 kb, and a small number of maxicircles each containing about 40 kb. Fractionation of kinetoplast DNA by equilibrium centrifugation in a CsCl-propidium dilodide gradient resolves it into three types of networks. Form I networks band at high density and contain minicircles which are covalently closed; form II networks band at low density and contain minicircles which are nicked or gapped; and replicating networks band at intermediate density and contain some minicircles of each type. Form I networks contain about 5000 minicircles; form II networks contain about 11,000; and replicating networks contain an intermediate number. When cells are pulse-labeled with ³H-thymidine, radioactivity in mitochondrial DNA is preferentially incorporated into replicating networks, but after a chase it appears first in form II networks and finally in form I. Examination of replicating networks by electron microscopy in the presence of ethidium bromide reveals that minicircles in the central region of the network are twisted and therefore covalently closed, whereas those in the peripheral region are not twisted and therefore must be nicked or gapped. The pulse-label is incorporated into the nicked or gapped minicircles of the replicating networks. These results indicate that replication of form I networks begins in peripheral minicircles and that progeny minicircles remain nicked or gapped. As replication proceeds, the size of the network increases, and the peripheral zone of nicked or gapped minicircles enlarges. Finally, when all minicircles have replicated, the network, now form II, is double the size of form I and contains only nicked or gapped minicircles. The final step in replication presumably includes both the cleavage of the network into two form I species and the covalent closure of all the minicircles.     

Nuclease TT1 from Thermus thermophilus HB8 has an endonuclease activity preferential to circular DNAs.

Homogeneously purified nuclease TT1 from Thermus thermophilus HB8 is known as an exonuclease to produce 5'-mononucleotides. Besides the exonuclease activity, nuclease TT1 also possesses endonuclease activity preferential to superhelical (form I) and single-stranded circular DNA. Although the rate of cleavage is slower than that of form I, covalently closed circular DNA (form I') is also cleaved. Form I DNA was nicked to yield relaxed circles (form II) first, and was then nicked at the opposite site to yield unit length linear DNA (form III) which was subsequently hydrolyzed to 5'-mononucleotides exonucleolytically. Both endo- and exo-nuclease activities co-migrate on polyacrylamide gels. The general properties of the endonuclease activity are very similar to those of the exonuclease activity. The temperature optimum for endonuclease activity was 85 degrees C. The pH-optimum was in pH-range from 7.5-9.1. The enzyme was active over a wide range of Mg2+ concentrations (2.5-125 mM), and was inhibited by EDTA. A linear substrate such as (dT)8 was a competitive inhibitor for this endonuclease activity.     

Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNAs.

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100-200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced by Hind II + III cleavage of phi80 plac DNA and phi80 plac DNA containing the Ll deletion (73 nucleotides in length). This heteroduplex incorporated twelve times as much iodine as the parent homoduplex fragments. Hence the technique could reveal the presence of a few (two or more) nonpaired cytosines, if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear lambdaplac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNA's revealed the absence of localized single-stranded regions.     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.     

A simple method for quantitative, semiquantitative, and qualitative assay of protein.

Aqueous cesium trichloroacetate permits the buoyant resolution of various RNAs and also of DNA at room temperature and neutral pH. Precipitate formation does not occur, under either native or denaturing conditions. The compositional buoyant density gradient was determined, and the buoyant densities of a variety of RNAs are presented. The buoyant densities increase in the order protein < DNA ? duplex RNA ? single-stranded RNA.     

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.