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Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoi...

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Autores principales: Kolesar, Jill E., Wang, Catherine Y., Taguchi, Yumiko V., Chou, Shih-Hsuan, Kaufman, Brett A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575812/
https://www.ncbi.nlm.nih.gov/pubmed/23275548
http://dx.doi.org/10.1093/nar/gks1324
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author Kolesar, Jill E.
Wang, Catherine Y.
Taguchi, Yumiko V.
Chou, Shih-Hsuan
Kaufman, Brett A.
author_facet Kolesar, Jill E.
Wang, Catherine Y.
Taguchi, Yumiko V.
Chou, Shih-Hsuan
Kaufman, Brett A.
author_sort Kolesar, Jill E.
collection PubMed
description The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion.
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spelling pubmed-35758122013-02-19 Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome Kolesar, Jill E. Wang, Catherine Y. Taguchi, Yumiko V. Chou, Shih-Hsuan Kaufman, Brett A. Nucleic Acids Res Methods Online The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion. Oxford University Press 2013-02 2012-12-25 /pmc/articles/PMC3575812/ /pubmed/23275548 http://dx.doi.org/10.1093/nar/gks1324 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.
spellingShingle Methods Online
Kolesar, Jill E.
Wang, Catherine Y.
Taguchi, Yumiko V.
Chou, Shih-Hsuan
Kaufman, Brett A.
Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title_full Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title_fullStr Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title_full_unstemmed Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title_short Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
title_sort two-dimensional intact mitochondrial dna agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575812/
https://www.ncbi.nlm.nih.gov/pubmed/23275548
http://dx.doi.org/10.1093/nar/gks1324
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