I. Studies of Closed Circular DNA : Physical and Biological Implications of Heterogeneity in the Topological Winding Number, α. II. The Structure of Virion SV40 DNA in Situ Examined by Chemical Modification with Dimethylsulfate

Author: Shure, Mavis

Year: 1979

Degree: Dissertation (Ph.D.)

Advisor: Vinograd, Jerome Rubin

Committee Member: Unknown, Unknown

Option: Biology

DOI: 10.7907/mn6t-xq83

Abstract

Methods for gel electrophoresis have been developed which permit the resolution of closed circular DNA molecules differing by unit values in their topological winding number, α. Relaxed or nonsupercoiled closed circular DNAs, native supercoiled DNAs, as well as DNAs having intermediate numbers of super-helical turns, can be resolved into sets of bands by one or more of the different electrophoresis conditions. In addition, a method based on theory (presented in Chapter IV) has been developed for the photographic quantitation of fluorescent substances. DNA stained with ethidium in agarose gels is used as an example. In the course of developing this method, it has been demonstrated that the empirical methods employed by other authors can give rise to large systematic errors.

The methods for electrophoresis, used in conjunction with the method for photographic quantitation of fluorescence, have permitted a quantitative examination of closed circular DNA. The results of such studies (presented in Chapters I, II, and III) are summarized below.

The limit product of the action of nicking-closing (N-C) enzyme on closed circular DNA is not a homogeneous species but rather a distribution. Each distribution has a mean degree of supercoiling of approximately zero. The individual species within the distributions differ by Δτ = ± 1, ± 2, etc., and the relative masses fit a Boltzmann distribution. It has also been demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for N-C 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.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA with N-C enzyme. Because native supercoiled DNA is heterogeneous with respect to α, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 ± 0.5 superhelical turns in 0.2 M NaCl and at 37°C, the conditions under which the enzymatic relaxations were performed. Under this same set of conditions, virion polyoma DNA contains 26 ± 1 superhelical turns, which is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as has SV40 DNA.

The average number of superhelical turns in SV40, 26, combined with the value, 21, for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 corresponds to the average number of superhelical turns within a nucleosome.

The superhelix densities determined by the band counting method have been compared with those determined by buoyant equilibrium in propidium diiodide (PDI)-CsCl gradients. A comparison of the values obtained by the two methods permits a calculation of an unwinding angle for ethidium. The mean value determined from experiments with SV40 DNA is 23 ± 3°.

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, α. All native closed circular DNAs examined, including the viral and intracellular forms of SY40 and polyoma DNAs, 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 enzyme on the respective DNAs. In addition, the distributions within the virion and the intracellular form I DNAs of SY40 were found to be indistinguishable. This was also found to be the case for the virion and the intracellular form I DNAs of polyoma.

In the cases of both SY 40 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 DN As poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution around 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 DN As would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in the N-C enzyme relaxed SY40 and polyoma DNAs.

Finally (see Chapter V), the structure of virion SY40 DNA in situ has been examined by chemical modification with dimethylsulfate (DMS). Virions are permeable to DMS and remain physically intact while the DNA (specifically, the bases adenine and guanine) within the virus particles becomes methylated. This approach permits the examination of specific protein-DNA interactions in the absence of artifacts of isolation and reconstitution.

A total length of approximately 3600 nucleotides (35% of the nucleotides in SV40 DNA) was scanned using this method. A total of 15 segments of the SV40 genome (obtained by kinasing 15 different 5' termini produced by treatment with different restriction endonucleases) was screened for decreases or increases in levels of methylation relative to naked SV40 DNA. The segments represented both strands of the DNA and were chosen to obtain samples representative of the entire genome, with a particular emphasis on fragments covering and surrounding the origin(s) of DNA replication and both late and early RNA transcription. No major or convincing differences were ever observed between purine patterns obtained after parallel treatment of corresponding samples derived from naked SV40 DNA which had been treated with DMS and from DNA within SV40 virions which had been treated with DMS.

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