In Vitro Studies of the Synthesis of Mitochondrial Deoxyribonucleic Acid

Citation

Tibbetts, Clark Joseph Bullock (1972) In Vitro Studies of the Synthesis of Mitochondrial Deoxyribonucleic Acid. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1XDN-S884. https://resolver.caltech.edu/CaltechTHESIS:09062016-133706695

Abstract

A DNA polymerase has been partially purified from the mitochondria of HeLa cells. Properties, including low levels of nuclease activity and a preference for native duplex DNA templates, were favorable for the study of in vitro DNA synthesis using circular duplex DNA templates. Initiation of DNA synthesis occurs predominately at single-strand scissions with covalent addition of nucleotides to the priming template strand. Centrifugation and electron microscopy have established that the template DNA strand ahead of the growing point is displaced rather than degraded. Hairpin structures are not formed in the course of DNA synthesis on duplex DNA templates. Studies with HeLa cell mitochondrial DNA template have indicated base composition-complement fidelity of the product of DNA synthesis as well as a preferential synthesis of DNA corresponding to the denser complement (in CsCl solution) of the template. This asymmetric synthesis of mitochondrial DNA appears to arise from a bias in the number of single-strand scissions sustained by the complementary strands.

DNA synthesis in isolated HeLa cell mitochondria has also been investigated. Product DNA appears in closed and nicked circular mitochondrial DNA. Preferential synthesis of DNA corresponding to the less dense complement (in CsCI solution) was observed.

The mode of action of the partially purified mitochondrial DNA polymerase and the asymmetric synthesis of mitochonarial DNA, observed in vitro and in situ, are discussed with regard to the results of recent studies of the replication of mitochondrial DNA in vivo.

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