DNA synthesis by isolated mitochondria: III. Characterization of D-loop DNA, a novel intermediate in mtDNA synthesis

Abstract

1. 1. We have previously shown that the main DNA product synthesized by isolated chick-liver mitochondria (called I∗) co-sediments with chick Component I (closed circular duplex form of mtDNA) in zone sedimentation and CsCl equilibrium gradients containing ethidium. On denaturation the radioactive DNA is released as short fragments (called E-strands). 2. 2. Heating of I∗ for 1 min at 95° in low salt results in the formation of non-radioactive 39-S DNA (Component I) and radioactive E-strands that sediment at about 9 S through neutral sucrose gradients (0.5 M NaCl). The majority of the E-strands electrophorese through an acrylamide gel in a single peak. 3. 3. DNA · DNA hybridization experiments show that denatured I∗ hybridizes as well with chick mtDNA as homologous chick mtDNA. The radioactive E-strands hybridize only with the L-strand of mtDNA (the strand with the lower equilibrium density in alkaline CsCl) and not with the complementary H-strand. The E-strands do not cross-hybridize with rat mtDNA, whereas 20–25 % of randomly labelled chick mtDNA will form an interspecific hybrid with rat mtDNA. 4. 4. The radioactive DNA in I∗ can be degraded more than 80 % by exonuclease III of Escherichia coli, showing that the E-strands are hydrogen-bonded over most of their length. 5. 5. I∗ can act as primer/template for DNA polymerase I of E. coli, leading to the incorporation of deoxyribonucleotides into a structure with the equilibrium density of a closed circular duplex in CsCl containing ethidium. 6. 6. The number of superhelical turns in I∗ was determined by band sedimentation through CsCl containing increasing concentrations of ethidium. The result suggests that I∗ contains about the same number of right-handed supertwists as Component I. 7. 7. The results are consistent with the model presented in Fig. 1. All radioactivity in I∗ is contained in the E-strand and the E-strand is a non-random fragment of the H-strand of mtDNA.