Mechanism of mitochondrial DNA replication in mouse L-cells: Topology of circular daughter molecules and dynamics of catenated oligomer formation

Abstract

Pulse-chase studies were performed, and the topology and specific activity of pulso-label in closed circular molecules determined, to investigate the mechanism by which newly segregated open circular daughter molecules are converted to the major stable form of mitochondrial DNA, D-mtDNA. These and previous results suggest the following model: newly segregated daughter molecules are first converted to closed circular molecules with a superhelix density of approximately zero. Watson-Crick turns are then removed by an unwinding mechanism to produce a stable intermediate with a superhelix density of ≈−0·03. Initiation of heavy-strand synthesis and polymerization of 450 nucleotides then occurs without further unwinding of the parental strands to yield the D-mtDNA molecule. The specific activity of pulse-label in catenated dimer mitochondrial DNA is 0·71 and 0·96 of that of monomer mitochondrial DNA after 60 and 150 minutes of chase, respectively. The topology of the interlocked monomers in pulse-labeled catenated dimers indicates that catenanes are not formed by aberrant segregation of daughter molecules. No linear intermediates in the segregation and closure of daughter molecules were detected. The simplest interpretation of these results is that monomers and catenated oligomers are in rapid equilibrium.