Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5′ protein

Mapping predicts that the mitochondrial genome of the liverwort Marchantia polymorpha exists as a circular molecule, although nearly all the mitochondrial DNA (mtDNA) is found as genome-sized and multigenomic molecules in linear and branched form. We used restriction enzymes with one recognition site per genome, end-specific exonucleases and pulsed-field gel electrophoresis (PFGE) to analyze the arrangement of genomic units and the terminal structure of the molecules. We find a head-to-tail arrangement in the concatemers and circular permutation in both the monomeric and multigenomic molecules. The termini contain covalently bound protein at the 5′ end and an open (unblocked) 3′ end. We find that the standard in-gel procedure used to prepare large DNA molecules for PFGE may introduce extraction artifacts leading to erroneous conclusions about the termini. These artifacts can be reduced by omitting high salt (high EDTA) and protease during mitochondrial lysis. Our results suggest that the mtDNA may use a T4 phage-like mechanism of replication and that the linear molecules may be due to strand breaks mediated by type II topoisomerase.