Structure and Properties of Closed Circular DNA

This thesis is concerned with the structure and properties of closed circular DNA. Part I of the thesis reports on experiments performed in this laboratory by the author and others to determine the structure of polyoma DNA component II. The following results were obtained: (1) Polyoma II is a ring-shaped duplex DNA molecule. (2) It is generated by introducing one single strand scission in polyoma I by the action of pancreatic DNAase or chemical reducing agents. (3) The sedimentation coefficient of polyoma II is insensitive to several single strand scissions. (4) Polyoma II, when not excessively attacked by pancreatic DNAase or chemical reducing agents, is infective. A publication is enclosed. The renaturation products of polynucleotide single strands obtained from nicked polyoma DNA were examined in Part II of the thesis. It was found that complementary, linear, single-stranded DNA can renature extensively. Preparations of single-stranded DNA containing more than 50% circular single strands renature to form two bands of DNA in CsCl buoyant density experiments. The buoyant density of one band is approximately that of native polyoma DNA and the buoyant density of the other is characteristic of denatured DNA. No evidence is found for any renaturation of complementary, circular, single-stranded DNA Part III of the thesis presents evidence indicating that nonnucleotide linkers are absent in both polyoma and øX174 DNA. This evidence was obtained by examining the exonuclease I digestion products of denatured1 pancreatic DNAase treated polyoma and øX174 DNA in alkaline CsCl velocity gradients. Part IV of the thesis contains a publication describing a dye-buoyant density method for the isolation and detection of closed circular DNA. The method is based on the reduced binding of the intercalating dye, ethidium bromide, by closed circular DNA. In an application of the method, it was found that HeLa cells contain, in addition to closed circular mitochondrial DNA of mean length 4.81 microns, a heterogeneous group of smaller DNA molecules which vary in size from 0.2 to 3.5 microns, and a paucidisperse group of multiples of the mitochondrial length. In addition, methodological results and data not presented in the publication are presented and discussed.