Abstract

DNA linked to an insoluble matrix has many potential applications. In some cases, it is highly desirable that the DNA be chemically unaltered, and for this reason, we have developed methods for topologically trapping circular DNAs on agarose. Open circular (oc) DNA containing at least one nick is readily trapped on agarose which has been heated or dissolved in sodium perchlorate to destroy secondary structure and then gelled by cooling or dialysis respectively. On the other hand, covalently closed circular (ccc) DNA of superhelix density −0.12 (PM2 DNA) is only poorly trapped unless first relaxed by topoisomerases or with the appropriate addition of an intercalating drug. When the oc DNA – agarose was used in a procedure for rapidly obtaining binding constants of drugs to DNA, the binding constant of ethidium was found to be considerably less than that expected. On addition of calf thymus topoisomerase to the binding-assay mixture, the ethidium-binding constant increased to the expected value. Thus, although free oc DNA is topologically unrestricted, oc DNA trapped in agarose must be rotationally constrained such that addition of ethidium introduced supercoils. The nature of these constraints is discussed with respect to the known structure of agarose bihelices.

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