PubMed · 4203840
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P Borst, L A Grivell. 1973. Mitochondrial nucleic acids.. https://doi.org/10.1016/s0300-9084(73)80032-x
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Results on electrophoretic capture of circular DNA in porous gels are reviewed. Processes which cause arrest of circular forms of DNA during electrophoresis can provide very efficient separation mechanism for the purification of plasmids and bacterial artificial chromosomes if the corresponding linear form is not trapped and therefore removed by the electric field. Two types of such topological traps have been proposed, impalement and lobster traps, and we here review the present experimental support for the existence of these two circle-specific mechanisms. Experiments designed to characterize the traps are discussed, regarding the concentration of the traps as well as their efficiency and capacity to trap both relaxed and supercoiled circular DNA. Studies of the dynamics of the capture process show that the average capture time is on the order of 10 s at 20 V/cm, by which time the circles have migrated several hundred micrometers and have passed hundreds of traps. We also review results on attempts to improve the capacity and efficiency of the trapping process by modification of the gels either by enzymatic treatment or by cogelation of neutral polymers.
The discovery of circular DNA, over 30 years ago, introduced an element of uneasiness in what had been, up to that point, the almost picture-perfect story of the elucidation of the molecular biology of heredity. If DNA indeed has the Watson-Crick right-handed helical secondary structure, then in circular DNA, thousands, or perhaps even millions of twists must be removed in each generation, and re-wound in the next generation. Although enzyme systems adequate for this task have long since been found and characterized, there have nevertheless arisen a number of proposals for alternative DNA structures in which the strands are topologically non-linked, so that they might separate during replication without having to be unwound. These structures have generally been put forth as theory only, and have been largely unaccompanied by experimental evidence to support their applicability to native DNA from living systems. Recently, however, a report has emerged suggesting that it might be possible to separate, intact, the individual single-stranded circular half-chromosomes which constitute the double-stranded circular chromosomes of certain plasmids. This would not be possible unless the chromosomes had one of the alternative, topologically non-linked structures. It is widely believed that after a half-century of worldwide DNA research, any significant change to the Watson-Crick structure is unlikely to stand up to scrutiny. Nevertheless, the present author has found that in many instances in which the behavior of circular duplex DNA is considered to be explicable only in terms of the topologically linked helical model, it is also possible to explain that same behavior in terms of a topologically non-linked model. It is necessary, in these instances, to make certain logical assumptions which cannot be conclusively proven at the present time. The author herein offers an example of one such instance, namely an examination of the behavior of circular duplex DNA in an alkaline titration experiment, where conformational changes in DNA are deduced from changes in its buoyant density at pH's between 7 and 14. These data have been explained in terms of topological linkage between the DNA strands, but they can also be explained without invoking any such topological linkage, provided that the above-mentioned logical assumptions can be accepted. The principles which emerge from this are applicable to other settings in which knowledge of the topology of DNA is critical to the understanding of observed phenomena.
Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination. We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types. We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events. Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K. lactis cells. Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule.