Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions.

The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of high concentrations of a variety of nonspecific polymers such as polyethylene glycol, Ficoll, bovine plasma albumin, or glycogen. Blunt-end ligation of small self-complementary oligodeoxyribonucleotides is also stimulated. The use of polyethylene glycol 6000 in such systems is characterized in some detail. Conditions are suggested which either greatly increase the rate of formation of and size of linear ligation products or which allow smaller but significant improvements in the amounts of circular ligation products.

Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.

In the presence of high concentrations of any of several types of macromolecules, DNA ligase preparations from rat liver nuclei or from Escherichia coli actively catalyze the blunt-end ligation of DNA. This is in contrast to the lack of activity on such substrates by these enzymes under conventional assay conditions. In addition, the previously established activity of T4 DNA ligase on blunt-ended molecules is greatly increased in the presence of high concentrations of macromolecules. Because such crowded solutions may well be a more adequate model for intracellular conditions than assays in dilute solutions, we suggest that blunt-end ligation may be a widely occurring reaction in vivo.

Conformational characteristics of deoxyribonucleic acid-butylamine complexes with C-type circular dichroism spectra. 1. An X-ray fiber diffraction study.

A set of complexes of calf thymus DNA and n-butylamine, covalently cross-linked to the DNA bases with CH2O, has been examined by X-ray diffraction. The attachment of this amine to DNA has been previously shown to result in a profound reduction of the rotational strength of the positive band above 260 nm in the circular dichroism (CD) spectrum in 20 mM NaCl, pH 7, without changing any of the properties which are characteristic of a native base-stacked duplex [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987]. In 20 mM NaCl, pH 7, substitution levels of 0-0.15 mol of amine/mol of nucleotide are sufficient to produce a family of CD spectra which range from the conservative one normally seen for protein-free DNA in this solvent to the nonconservative one ascribed to the C form of DNA [Hanlon, S., Brudno, S., Wu, T. T., & Wolf, B. (1975) Biochemistry 14, 1648-1660]. Fibers of the DNA X amine complexes at 79% relative humidity (rh) and 33% rh do indeed show C-type X-ray patterns whereas the controls exhibit A forms under the same conditions. The same preparations, however, exhibit only B patterns when the fibers are examined at 98% rh and in the wet fiber form. Although we cannot rule out the possibility that molecular interactions in the fiber have imposed conformational restraints on the DNA structure that are not present in solution, these present results suggest that the conformation of DNA giving rise to the "C" CD spectrum is a variant of the B form.

A RNA.DNA hybrid that can adopt two conformations: an x-ray diffraction study of poly(rA).poly(dT) in concentrated solution or in fibers.

The RNA.DNA hybrid poly(rA).poly(dT) can adopt two conformations, depending upon its degree of hydration. Fibers yield a conventional A' RNA-like pattern at 79% relative humidity. In contrast, under highly solvated conditions this material yields an x-ray diffraction pattern with striking similarities to that of B DNA and clearly different from the RNA-like diffraction patterns previously obtained from fibers of other hybrids. A structural model is proposed for the solution form of the hybrid that has several similarities to models proposed for B DNA. The hybrid model accommodates the 2'-hydroxyl groups of the poly(rA) strand in intrachain hydrogen bonds to the adjacent ribose moieties.

Nonuniform backbone conformation of deoxyribonucleic acid indicated by phosphorus-31 nuclear magnetic resonance chemical shift anisotropy.

31P nuclear magnetic resonance of highly oriented DNA fibers has been observed for three different conformations, namely, the A, B, and C forms of DNA. At a parallel orientation of the fiber axis with respect to the magnetic field, DNA fibers in both the A and B forms exhibit a single, abnormally broad resonance; in contrast, fibers in the C form show almost the full span of the chemical shift anisotropy (170 ppm). The spectra of the fibers oriented perpendicular indicate that the DNA molecules undergo a considerable rotational motion about the helical axis, with a rate of greater than 2 x 10(3) s-1 for the B-form DNA. Theoretical considerations indicate that the 31P chemical shift data for the B-form DNA fibers are consistent with the atomic coordinates of the phosphodiester group proposed by Langridge et al. [Langridge, R., Wilson, H. R. Hooper, C. W., Wilkins, M. H. F., & Hamilton, L. D. (1960) J. Mol. Biol. 2, 19--37] but not with the corresponding coordinates proposed by Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Coomun. 47, 1504--1509], and also lead to the conclusion that the phosphodiester orientation must vary significantly along the DNA molecule. The latter result suggests that DNA has significant variations in its backbone conformation along the molecule.

Helical parameters of DNA do not change when DNA fibers are wetted: X-ray diffraction study.

We have measured the helical parameters of DNA in concentrated solutions by x-ray fiber diffraction methods. Fibers of the sodium salt of DNA were swollen with water within capillaries; the capillary served to limit water uptake, slowing dissolution. Samples containing up to 80% water gave essentially a B-form diffraction pattern and had virtually the same helical parameters [9.91 base pairs per turn (SD = 0.14); 3.34 A axial rise per residue (SD = 0.019)] as did the initial fibers [9.95 base pairs per turn (SD = 0.15); 3.33 A axial rise per residue (SD = 0.015)]. Hence, under highly solvated conditions in which the interactions between molecules should be greatly decreased, DNA maintains its classical B-form structure.

Hybrid isolation by recovery of RNA-DNA hybrids from agar using S1 nuclease.

A method for recovering RNA-DNA hybrids from agar employing a single strand specific nuclease is described. The procedure is suitable for large scale isolations, and immobilization of the DNA in agar prior to hybridization allows a high yield of hybrid without interference by DNA reannealing.