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.
Highlights
Analysis of human heart mitochondrial DNA by electron microscopy and agarose gel electrophoresis revealed a complete absence of the -type replication intermediates seen abundantly in mtDNA from all other tissues
In the present work we addressed further the nature and significance of the junctional molecules in human heart mtDNA using a combination of one- and two-dimensional agarose gel electrophoresis (AGE), transmission electron microscopy (TEM), and enzymatic treatments
MtDNA Replication Intermediates Differ between Tissues—We set out initially to characterize the forms of mtDNA and its replication intermediates present in different human tissues
Summary
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. Subsequent to this, a novel mechanism of DNA replication was proposed based on further electron microscopy and end mapping [11,12,13,14] This strand displacement model envisaged a single unidirectional origin for synthesis of the leading (heavy) strand (OH), which proceeds approximately two-thirds of the way around the genome until the lagging (light) strand origin (OL) is exposed on the displaced parental heavy strand. The pattern of mtDNA replication intermediates detected in the tissue of mice transgenic for either of two proteins involved in mtDNA maintenance, mitochondrial transcription factor A (TFAM) and Twinkle, shows a substantial increase in junctional forms
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