Pulse-label analysis and mapping of the two terminal regions of asynchronous complementary strand replication of mitochondrial DNA in transformed hamster cells

Abstract

In vivo pulse-chase radioactive labeling studies were performed to localize within the physical map of C 13 B 4 hamster mtDNA † † Abbreviations used: mtDNA, mitochondrial DNA; for all other abbreviations, see accompanying paper (Nass, 1980). the two terminal regions of heavy and light complementary strand synthesis. These terminal segments have been defined operationally as that region on the H- and L-strand that is synthesized last. mtDNA of monolayer cultures was pulsed with [ 3H]thymidine for a minimum period of 10 minutes, which is about one-tenth of one round of mtDNA synthesis, followed by chase periods of up to 120 minutes. The properties of the labeled closed circular replicative intermediates E-mtDNA, C-mtDNA and D-mtDNA were analyzed in CsCl PrI 2 gradients and in neutral sucrose velocity and alkaline CsCl gradients. Both terminally labeled α and β daughter molecules were found to pass through the E-mtDNA stage. Sensitivity of C 13 B 4 mtDNA to alkali and ribonuclease A indicated the presence of covalently linked ribonucleotides. The distribution (specific activity) of pulse-chase radioactivity relative to uniform label was followed in electrophoretically separated HpaII + HinIII and HpaI restriction fragments (freed of 7 S initiation sequences) and corrected for thymine content. The strand specificity of the pulse-label was determined by hybridization of restriction fragments with H- and L-strands of mtDNA. The kinetic data agree precisely with electron microscopic determinations of H- and L-strand origins at respective genome positions of 0 and 67 ± 3, which are located on HpaII + HindIII fragments 9 and 6, respectively (Nass, 1980). The two terminal regions are within the predicted genome sector between about 67 and 100/0 map units; the highest terminal pulse-chase radioactivity extends within 5 to 15% of the genome's length behind each origin. The kinetics of early labeling events were found to differ at the two termini. The evidence indicates that the majority of L-strand initiation/termination sites are in the region near map position 67 ± 3, and confirms the highly asynchronous replication mechanism of this DNA.