DNA and RNA can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex regions. By assaying at different pHs and introducing a heating/cooling cycle, a great many physicochemical aspects of DNA and RNA can be studied avoiding the use of radiolabels, and often giving information not otherwise readily obtainable. Studies are described on duplex DNA which involve measurement of extinction coefficients, cross-linking by chemicals, Cot curve analysis as well as estimation of drug-DNA binding constants. The assays can be adapted to investigate multi-stranded nucleic acid structures. The use of covalently closed circular DNA also allows rapid and extremely sensitive measurements of nicking caused by irradiation or drugs.
The DNA polymer d(GC)n . d(GC)n can undergo a transition from the usual right-handed 10.4 base pairs (bp) per turn B form to a novel left-handed 12 bp per turn Z form in response to altered environmental conditions. Several other alternating purine-pyrimidine DNA polymers with modified bases have been shown to undergo transitions from B to Z conformations, with varying degrees of difficulty. We report here that the unmodified DNA polymer d(TG)n . d(CA)n readily undergoes a transition to a Z conformation when subjected to unwinding torsional stress in ionic conditions that are close to physiological. By using a two-dimensional gel electrophoresis system, we have determined both the critical free energy of supercoiling that is required to initiate the transition and the free energy of supercoiling that is required to maintain this polymer in the Z form.
Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColEl, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by Ar = i1, i42, etc., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution.Nicking-closing (N-C) activities that alter the topological winding number (a) of closed circular DNA occur widely in nature (1-5). The topological winding number is the number of revolutions that one strand makes about the other if the molecule is constrained to lie in a plane. The activities have been demonstrated to be enzymatic with proteins from Escherichia coli (6), mouse (7), and human (8) cells in culture. The N-C enzyme from mouse LA9 cell nuclei, purified to homogeneity in good yield, is a major constituent of chromatin and accounts for about 1% of the total protein (H-P.Vosberg and J. Vinograd, unpublished work). It is similar to other eukaryotic N-C enzymes in its ability to relax both positive and negative superhelical turns. A probable in vivo role for the enzyme is to provide the transient swivels required for DNA replication. Such swivels may also be required in transcription, and in the condensation and decondensation of chromatin.In this study we have examined the limit product of the action of N-C enzyme on several closed circular DNAs by gel electrophoresis. Under appropriate analytical conditions, the limit product separates into a set of species differing in topological winding number. Individual species, isolated from the set, regenerate the original distribution upon incubation with the N-C enzyme. We view the foregoing as the consequence of four necessary events: nicking of the DNA, relaxation, random rotation about the swivel, and closure (Fig. 1). A set of species is also found when E. colh polynucleotide ligase is used to close a nicked circular DNA (9, 10). It is shown here that distribution of products obtained with ligase is indistinguishable from that obtained with N-C enzyme when the incubation conditions are the same for both reactions.The relative masses of the species, when plotted against a, fall on a Gaussian curve. Such a curve is anticipated for a Boltzmann distribution, when the energy of supercoiling is Abbreviations: N-C, nicking-closing; EtdBr, ethidium bromide.proportional to the square (11) of the degree of supercoiling. The similarity of the products obtained with two different enzyme systems and the further similarities of the values of the free energy of supercoiling obtained...
Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs. The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images
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