Studies of the replicative intermediates and of the structure of animal cell mitochondrial DNA

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Studies on the replication and structure of animal cell mitDNA are reported in the four sections of this thesis.

Part I: Density Labeling of HeLa mitDNA with 5-Bromodeoxyuridine: Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/mi. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.

Part II: The Isolation of Complementary Strands of mitDNA and the Mapping of the rDNA and tRNA's on the L Strand Relative to the Origin of Replication: The alkali lability of mitDNA makes the preparation of intact separated strands for electron microscope mapping studies difficult. Even at pH 11.6 (the minimum pH required for strand separation of singly nicked HeLa mitDNA) the strands suffered an average of one additional nick per strand during the alkaline buoyant CsCl banding. Covalently closed mitDNA did not nick under these conditions. The rapid nicking of the BrUra-labeled strands enabled preparations of L strands containing 30 wt% single stranded circles to be obtained from covalently closed BrUra hybrid mitDNA.

The relative positions of the ribosomal RNA gene complement (rDNA) and the tRNA sites on the L strand relative to the origin of replication (7S DNA) were determined by hybrid mapping in the electron microscope using the above L strand preparation. The center of the rDNA is located almost directly opposite (0.5 G) the 7S DNA, and the L3 tRNA site is very near to one end of the 7S DNA.

Part III: MitDNA Replicative Intermediates: Isolation by Benzoylated DEAE-Cellulose Chromatography and Enzymatic Analysis of Structure: Replicative intermediates of LA9 mitDNA were partially purified from clean duplex DNA by virtue of the binding of their single stranded regions to benzoylated DEAE-cellulose. The purification was incomplete because the clean duplex DNA elutes as though a portion of the molecules contain denatured regions (or regions which denature in response to the column environment). Similar results were obtained with closed and nicked PM2 viral DNA. SV40 viral DNA eluted normally.

These enriched preparations of replicative intermediates and the clean duplex DNA free of replicating forms were subjected to enzymatic analysis with T4 DNA polymerase. The results show that: (1) the nicks in the upper band clean duplex DNA are not preferentially located in either strand; (2) the gapped molecules in the upper band have a greater overall deficiency of L than H strand DNA by a ratio of 2.5 to 1 (implying that these molecules probably represent displaced H strands with incomplete complement synthesis); and (3) the 7S DNA in the lower band D-loop forms is not measurably extended by the enzyme, although label was incorporated (suggesting that a superhelix restriction on the further extension of the D-loop exists in isolated mitDNA). Attempts to covalently close the upper band clean duplex DNA with combinations of E. coli exonuclease III, polymerase I, and ligase, as well as T4 polymerase, were unsuccessful.

Part IV: A Method for the Detection of Ribonucleotides in mitDNA: A method for the detection of ribonucleotides in mitDNA has been partially developed. The procedure involves alkaline hydrolysis to expose ribonucleotides at the 3' ends of mitDNA fragments, removal of terminal phosphates with alkaline phosphatase, labeling by oxidation-reduction with NaIO4-3H-NaBH4, removal of unincorporated 3H counts by extensive dialysis, spleen acid DNase II and spleen exonuclease digestion to ribonucleoside trialcohols and deoxynucleoside-3'-monophosphates, removal of the latter on DEAE-cellulose, and identification of labeled ribonucleoside trialcohols by thin layer chromatography and fluorography. The feasibility of the analysis is demonstrated, but attempts to label mitDNA were unsuccessful due to the unreliability of commercial preparations of 3H-NaBH4 of the high specific activity required for these experiments. The method is reported here in the event that high specific activity 3H-NaBH4 becomes available.