Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH
01 Jun 1984-International Journal of Biological Macromolecules (Elsevier)-Vol. 6, Iss: 3, pp 152-154
TL;DR: Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization, and gel electrophoretic analysis revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which was designated as RF I d.
Abstract: Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I d . This faster-moving form is cleaved by the restriction endonuclease Bam HI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by Bam HI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I d as suggested previously.
TL;DR: It is proposed that form V consists of right-handed Watson-Crick type double-helices which are compensated by an equal number of left-handed duplex turns and negative supercoils, giving rise to a two-stranded complex that has well-defined physico-chemical properties.
Abstract: Complementary circular single strands of plasmid PβG or bacteriophage PM2 DNA but not of single-stranded φX174 DNA associate under hybridisation conditions, giving rise to a two-stranded complex. This DNA, which we call form V, has well-defined physico-chemical properties. It sediments as a sharp peak in neutral sucrose gradients; its electrophoretic mobility in agarose gels is between that of covalently closed (form I) and denatured DNA. In the electron microscope form V appears as highly folded duplex molecules indistinguishable from form I. However, increasing concentrations of ethidium bromide which lead to relaxation and recoiling of form I DNA have no comparable effect upon form V. At 260 nm form V PβG DNA has a hypochromicity of 18.6%, as compared to 23.4% in the case of PβG form II DNA and 10.5% in the case of single-stranded φX174 DNA. The thermal melting of form V is non-cooperative with gradual increase in absorbance similar to that of single-stranded DNA. The circular dichroism spectrum of form V DNA differs from that of form I, circular nicked (form II) and single-stranded φX174 DNA in that it shows a negative band at 295 nm and a shift for the main positive band from 273 to 266 nm. We propose that form V consists of right-handed Watson-Crick type double-helices which are compensated by an equal number of left-handed duplex turns and negative supercoils. Wo cannot decide whether left-handed duplex turns are stabilised by base-stacking and hydrogen bonding, as for example in the models described by Rodley et al. (1976) or Sasisekharan & Pattabiraman (1976), or whether they are merely compensatory turns without inherent stability.
TL;DR: It is concluded that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.
Abstract: Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.
TL;DR: 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 and it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction.
Abstract: 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.