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

Abstract

synopsisThe 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 sedimentation 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.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 accord 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 Xbzbsc DNA circles. Possible explanations for some of the above results are discussed.A corresponding result is observed in the intrinsic viscosity vs. pH curve.

Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities

“…232 All DNA samples were dialyzed against BE buffer except when treatment with polynucleotide ligase was performed, in which case dialysis was against DNase I buffer (0.015 M NaCl, 0.01 M Tris, 1 mM EDTA, pH 8.1). 23 When necessary, DNA samples were concentrated by pelleting in the SW50.1 rotor at 45 or 50 Krpm near 10°C and resuspended in approximately 0.2 ml of the supernatant. 24 Anatyticat uttAacentiu9gationY.…”

“…Analytical band sedimentation33 was performed in a Beckman Model E ultracentrifuge with photoelectric scanning system as described previously. 23,24 Neutral and alkaline sedimentation solvents, containing CsCl at a density near 1.35 g/ml, have the compositions given earlier.34 Sedimentation coefficients were evaluated by a least squares analysis procedure in which seriously deviating data points are eliminated.23 30 mm centerpieces were used with the Xb2b5c and T5 DNA's to reduce the effects of concentration-dependent sedimentation.…”

Superhelix density heterogeneity in closed circular intracellular PM2 DNA

“…'~ All sedimentatioii coefficients reported are uncorrected values at infinite dilution so, evaluated as previously dcscribcd. 16 At thr DNA concentrations employed in these studies, there is no question of the spreading and skewing observed in som(' of the bands (e.g., Figurcb 3) arising from conccntration dependence. Maximal concentrations are approximately cqual in both strongly spreading and nonsprcading bands in most instances.…”

“…Band sedimentation velocity experiments were performed in a Beckman Model E ultracentrifuge equipped with photoelectric scanning system as previously described. 16 All experiments (with the exception of one each at 15°C and at 23.5"C) were carried out at 25°C. Alkaline sedimentation solvent consisted of 2.83 M CsC1, 0.10 M KOH, 0.001 M EDTA while neutral solvent contained 2.83 M CsCl, 0.01 M tris, 0.001 M EDTA, pH 8.1, density near 1.33 g/ml.…”

Endonucleolytic incision of x-irradiated deoxyribonucleic acid by extracts of Escherichia coli.

“…OX-174 RFI or PM2 I was x-irradiated in Tris-EDTA buffer for a series of doses, adjusted to pH 12.2 or 11.9, respectively, neutralized, and analyzed for the percent remaining RFI or PM2 I on low-salt, neutral sucrose gradients [kX-174 RFI and PM2 I are reversibly renatured under these conditions (28,32) ]. In either DNA sample, about one alkali-labile site was detected for every single-strand break observed under neutral conditions.…”