Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks

Abstract

Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals.

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…Overexpression of Twinkle was shown to increase the presence of Holliday junction type structures in heart mtDNA of transgenic mice (45). These findings coupled with observations that the major strand exchange recombination protein Rad51 and the related HR proteins Rad51C and Xrcc3 are found in human mitochondria (46) suggest that HR repair of mitochondrial double strand breaks is likely to be important for maintaining genome stability in the organelle.…”

“…Electron microscopy and agarose gel analyses have provided evidence that human heart mtDNA contains multimeric junctional DNA complexes, including four-way and three-way junctions typically associated with replication forks or intermediates of HR repair (45). Overexpression of Twinkle was shown to increase the presence of Holliday junction type structures in heart mtDNA of transgenic mice (45).…”

Mgm101 Is a Rad52-related Protein Required for Mitochondrial DNA Recombination

“…60 and 61). Notably, recent electron microscopic studies have revealed the presence of abundant four-way and three-way junctions suggestive of recombination intermediates in the oxyradical-rich human adult heart and brain mitochondria (62)(63)(64). A low level of recombination, which is probably limited between intranucleoidal mtDNA molecules (65), may be in place in animal mitochondria from specific tissues as an effective mechanism for mutational clearance while minimizing the invasion by selfish DNAs (66).…”

The road to rack and ruin: selecting deleterious mitochondrial DNA variants

“…Moreover, in a laboratory strain of mice there was no evidence of intermolecular mtDNA recombination in the germ-line over the course of 50 generations [42]. An ability to resolve recombination junctions might be required at the end of the replication cycle as found for bacteria [43], and be necessary to regulate the networks of mtDNA molecules that form in adult human heart [44]. It is unlikely that mitochondria can create such DNA structures and yet be unable to resolve or otherwise process them.…”