On the origin of tertiary turns in covalently closed double-stranded cyclic DNA.

Abstract

At certain stages of the life cycle, many viral DNA's occur as duplexes (for a review, see ref. 1). Physicochemical studies by Vinograd and co-workers have shown that such molecules contain tertiary turns topologically locked into the molecules,l 2 as a consequence of which the molecules are more compact hydrodynamically and hence have sedimentation coefficients higher than those for molecules without tertiary turns. When examined under the electron microscope by Kleinschmidt method, such molecules appear in agreement with the notion that the molecules contain tertiary turns; hence the name twisted DNA or supercoil is frequently used. If one single-chain scission is introduced in a DNA, a swivel is provided. By rotating around the single bond opposite to the point of the single-chain scission, the molecule may release its tertiary turns. This is accompanied by a decrease in sedimentation coefficient, as well as other changes in the physicochemical properties of the molecule. For the purpose of simplicity, we shall refer to double-stranded molecules as covalently DNA or closed circles, and double-stranded molecules with one or more single-chain scissions as free-swiveling DNA or simply as cyclic Inasmuch as all the DNA's thus far isolated from nature appear to be twisted, the origin of the tertiary turns in such molecules is of interest. Several hypotheses have been suggested: (1) The molecule was partially unwound (i.e., a segment of denatured region was present) at the time of final closure. Since the total winding number of a is a topological invariant, I the winding of the unwound region associated with the base-pairing after closure necessarily introduces tertiary turns of the opposite sense into the molecule. One reason for such an unwound region might be to accommodate the closing enzyme.1 (2) The number of base-pairs per Watson-Crick helical turn of a helix in vivo is different from the number of base-pairs per turn of the helix in the purified DNA. The change of the number of Watson-Crick helical turns of the results in tertiary turns in the molecule. (3) The molecule in vivo at the time of final closure is wound on a protein core; the removal of the core during the purification of the produces tertiary turns.2 4 The work of Gellert with Escherichia coli extracts showed that it was possible to form X from X in vitro.5 It was later shown that the active ingredients in the extracts were a polynucleotide-joining enzyme and its cofactor, diphosphopyridine nucleotide (DPN).1-9 In the presence of the cofactor, the enzyme can link two polynucleotide chains through a 3'5' phosphodiester bond, provided that the 3' hydroxyl and the 5' phosphate ends of