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Increased negative supercoiling of mtDNA in TOP1mt knockout mice and presence of topoisomerases IIα and IIβ in vertebrate mitochondria.

Zhang H, Zhang YW, Yasukawa T, Dalla Rosa I, Khiati S, Pommier Y - Nucleic Acids Res. (2014)

Bottom Line: Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology.Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates.The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology and Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA pommier@nih.gov.

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Top2 cleavage complexes in mtDNA. (A) Top2 cleavage complexes in mitochondria from MCF7 cells treated with etoposide. Mitochondrial lysates were fractioned by CsCl gradient. Three consecutive DNA-containing fractions of each sample were collected and immunoblotted with Top2 antibody (ICE bioassay). (B) Cleavage sites in mtDNA from TOP1mt TOP2β double-knockout MEFs and their wild-type (WT) counterpart treated with etoposide. PL-PCR was used to visualize mtDNA fragments. Ref: non-specific band used as reference. (C) Map summarizing the etoposide-induced cleavage sites shown in panel (B) (arrows). (D) Characterization of the Top2α site at nucleotide positions H15 426 and L16 038 observed under normal growth conditions. Both sites were characterized with respect to heat-reversibility (R) (65°C for 5 min), merbarone-sensitivity (M) (200 μM for 4 h) and regeneration by addition of purified human Top2α to mtDNA from merbarone-treated cells. (E) Schematic drawing showing the notable position of the H15 426 and L16 038 sites. The 7S DNA in the D-loop is shown as dashed line.
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Figure 5: Top2 cleavage complexes in mtDNA. (A) Top2 cleavage complexes in mitochondria from MCF7 cells treated with etoposide. Mitochondrial lysates were fractioned by CsCl gradient. Three consecutive DNA-containing fractions of each sample were collected and immunoblotted with Top2 antibody (ICE bioassay). (B) Cleavage sites in mtDNA from TOP1mt TOP2β double-knockout MEFs and their wild-type (WT) counterpart treated with etoposide. PL-PCR was used to visualize mtDNA fragments. Ref: non-specific band used as reference. (C) Map summarizing the etoposide-induced cleavage sites shown in panel (B) (arrows). (D) Characterization of the Top2α site at nucleotide positions H15 426 and L16 038 observed under normal growth conditions. Both sites were characterized with respect to heat-reversibility (R) (65°C for 5 min), merbarone-sensitivity (M) (200 μM for 4 h) and regeneration by addition of purified human Top2α to mtDNA from merbarone-treated cells. (E) Schematic drawing showing the notable position of the H15 426 and L16 038 sites. The 7S DNA in the D-loop is shown as dashed line.

Mentions: To detect Top2 activity in mitochondria, we took advantage of the fact that type II topoisomerases cleave the DNA backbone by covalent attachment to the 5′-ends of the break, and that these transient catalytic intermediates, which are referred to as cleavage complexes (2,3) can be trapped by etoposide, a selective Top2α–Top2β poison widely used for cancer treatment (3,35,36). Consistent with the presence of Top2α and Top2β in mitochondria, treatment of purified mitochondria from MCF-7 cells with etoposide generated Top2-DNA complexes, as measured by the ICE bioassay (22,23) (Figure 5A).


Increased negative supercoiling of mtDNA in TOP1mt knockout mice and presence of topoisomerases IIα and IIβ in vertebrate mitochondria.

Zhang H, Zhang YW, Yasukawa T, Dalla Rosa I, Khiati S, Pommier Y - Nucleic Acids Res. (2014)

Top2 cleavage complexes in mtDNA. (A) Top2 cleavage complexes in mitochondria from MCF7 cells treated with etoposide. Mitochondrial lysates were fractioned by CsCl gradient. Three consecutive DNA-containing fractions of each sample were collected and immunoblotted with Top2 antibody (ICE bioassay). (B) Cleavage sites in mtDNA from TOP1mt TOP2β double-knockout MEFs and their wild-type (WT) counterpart treated with etoposide. PL-PCR was used to visualize mtDNA fragments. Ref: non-specific band used as reference. (C) Map summarizing the etoposide-induced cleavage sites shown in panel (B) (arrows). (D) Characterization of the Top2α site at nucleotide positions H15 426 and L16 038 observed under normal growth conditions. Both sites were characterized with respect to heat-reversibility (R) (65°C for 5 min), merbarone-sensitivity (M) (200 μM for 4 h) and regeneration by addition of purified human Top2α to mtDNA from merbarone-treated cells. (E) Schematic drawing showing the notable position of the H15 426 and L16 038 sites. The 7S DNA in the D-loop is shown as dashed line.
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Figure 5: Top2 cleavage complexes in mtDNA. (A) Top2 cleavage complexes in mitochondria from MCF7 cells treated with etoposide. Mitochondrial lysates were fractioned by CsCl gradient. Three consecutive DNA-containing fractions of each sample were collected and immunoblotted with Top2 antibody (ICE bioassay). (B) Cleavage sites in mtDNA from TOP1mt TOP2β double-knockout MEFs and their wild-type (WT) counterpart treated with etoposide. PL-PCR was used to visualize mtDNA fragments. Ref: non-specific band used as reference. (C) Map summarizing the etoposide-induced cleavage sites shown in panel (B) (arrows). (D) Characterization of the Top2α site at nucleotide positions H15 426 and L16 038 observed under normal growth conditions. Both sites were characterized with respect to heat-reversibility (R) (65°C for 5 min), merbarone-sensitivity (M) (200 μM for 4 h) and regeneration by addition of purified human Top2α to mtDNA from merbarone-treated cells. (E) Schematic drawing showing the notable position of the H15 426 and L16 038 sites. The 7S DNA in the D-loop is shown as dashed line.
Mentions: To detect Top2 activity in mitochondria, we took advantage of the fact that type II topoisomerases cleave the DNA backbone by covalent attachment to the 5′-ends of the break, and that these transient catalytic intermediates, which are referred to as cleavage complexes (2,3) can be trapped by etoposide, a selective Top2α–Top2β poison widely used for cancer treatment (3,35,36). Consistent with the presence of Top2α and Top2β in mitochondria, treatment of purified mitochondria from MCF-7 cells with etoposide generated Top2-DNA complexes, as measured by the ICE bioassay (22,23) (Figure 5A).

Bottom Line: Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology.Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates.The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology and Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA pommier@nih.gov.

Show MeSH
Related in: MedlinePlus