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Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element.

Jemt E, Persson Ö, Shi Y, Mehmedovic M, Uhler JP, Dávila López M, Freyer C, Gustafsson CM, Samuelsson T, Falkenberg M - Nucleic Acids Res. (2015)

Bottom Line: The other motif, here denoted coreTAS, is at the D-loop 3'-end.Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication.Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden.

No MeSH data available.


Recovery of mtDNA reduces termination of HSP transcription at coreTAS and creates longer antisense D-loop transcripts. (A) Quantification of mtDNA levels of ddC-treated cells. The graph shows the percentage of mtDNA levels compared to nuclear DNA levels. For details about DNA primers, see ‘Materials and Methods’ section. One representative experiment is shown. (B) Southern blot analysis as described in ‘Materials and Methods’ section of mtDNA from ddC treated (+) or untreated (−) cells using strand-specific probes that detect the D-loop region. The ratio of 7S DNA to mtDNA (%) is also shown. Lanes 1 and 2, untreated/ddC treated cells for 3 days; lanes 3 and 4, untreated/ddC-treated cells recovered for 2 days; and lanes 5 and 6, untreated/ddC-treated cells recovered for 5 days. (C) Schematic presentation of the two strand-specific probes (situated inside or outside the D-loop region) that were used for the experiment in (D). (D) Northern blot analysis of total RNA from ddC treated (+) or untreated (−) cells. Probes used are described in ‘Materials and Methods’ section and are depicted in (C). The levels of antisense transcript relative CYTB (%) are indicated. The experiments were repeated at least three times and one representative experiment is shown. The presented northern blots for the inside and outside probes represent two individual gels that have been run in parallel. For abbreviations, see legend to Figure 1.
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Figure 4: Recovery of mtDNA reduces termination of HSP transcription at coreTAS and creates longer antisense D-loop transcripts. (A) Quantification of mtDNA levels of ddC-treated cells. The graph shows the percentage of mtDNA levels compared to nuclear DNA levels. For details about DNA primers, see ‘Materials and Methods’ section. One representative experiment is shown. (B) Southern blot analysis as described in ‘Materials and Methods’ section of mtDNA from ddC treated (+) or untreated (−) cells using strand-specific probes that detect the D-loop region. The ratio of 7S DNA to mtDNA (%) is also shown. Lanes 1 and 2, untreated/ddC treated cells for 3 days; lanes 3 and 4, untreated/ddC-treated cells recovered for 2 days; and lanes 5 and 6, untreated/ddC-treated cells recovered for 5 days. (C) Schematic presentation of the two strand-specific probes (situated inside or outside the D-loop region) that were used for the experiment in (D). (D) Northern blot analysis of total RNA from ddC treated (+) or untreated (−) cells. Probes used are described in ‘Materials and Methods’ section and are depicted in (C). The levels of antisense transcript relative CYTB (%) are indicated. The experiments were repeated at least three times and one representative experiment is shown. The presented northern blots for the inside and outside probes represent two individual gels that have been run in parallel. For abbreviations, see legend to Figure 1.

Mentions: As shown in Figure 4A, three days of ddC treatment led to depletion of the full-length mtDNA (to about 20%). After the ddC had been removed from the media, five days of recovery were necessary to obtain a normal level of mtDNA. Southern blotting of untreated and ddC treated cells revealed that the ratio of 7S DNA to mtDNA was changed in the ddC treated cells compared to untreated controls. Three days of ddC treatment caused a strong reduction, but did not completely abolish 7S DNA levels (Figure 4B, lane 2). After removal of ddC, we saw a gradual recovery of 7S DNA levels relative full-length mtDNA (Figure 4B). This result is in agreement with the previous finding that the premature DNA replication termination event is less frequent after mtDNA depletion (50).


Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element.

Jemt E, Persson Ö, Shi Y, Mehmedovic M, Uhler JP, Dávila López M, Freyer C, Gustafsson CM, Samuelsson T, Falkenberg M - Nucleic Acids Res. (2015)

Recovery of mtDNA reduces termination of HSP transcription at coreTAS and creates longer antisense D-loop transcripts. (A) Quantification of mtDNA levels of ddC-treated cells. The graph shows the percentage of mtDNA levels compared to nuclear DNA levels. For details about DNA primers, see ‘Materials and Methods’ section. One representative experiment is shown. (B) Southern blot analysis as described in ‘Materials and Methods’ section of mtDNA from ddC treated (+) or untreated (−) cells using strand-specific probes that detect the D-loop region. The ratio of 7S DNA to mtDNA (%) is also shown. Lanes 1 and 2, untreated/ddC treated cells for 3 days; lanes 3 and 4, untreated/ddC-treated cells recovered for 2 days; and lanes 5 and 6, untreated/ddC-treated cells recovered for 5 days. (C) Schematic presentation of the two strand-specific probes (situated inside or outside the D-loop region) that were used for the experiment in (D). (D) Northern blot analysis of total RNA from ddC treated (+) or untreated (−) cells. Probes used are described in ‘Materials and Methods’ section and are depicted in (C). The levels of antisense transcript relative CYTB (%) are indicated. The experiments were repeated at least three times and one representative experiment is shown. The presented northern blots for the inside and outside probes represent two individual gels that have been run in parallel. For abbreviations, see legend to Figure 1.
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Figure 4: Recovery of mtDNA reduces termination of HSP transcription at coreTAS and creates longer antisense D-loop transcripts. (A) Quantification of mtDNA levels of ddC-treated cells. The graph shows the percentage of mtDNA levels compared to nuclear DNA levels. For details about DNA primers, see ‘Materials and Methods’ section. One representative experiment is shown. (B) Southern blot analysis as described in ‘Materials and Methods’ section of mtDNA from ddC treated (+) or untreated (−) cells using strand-specific probes that detect the D-loop region. The ratio of 7S DNA to mtDNA (%) is also shown. Lanes 1 and 2, untreated/ddC treated cells for 3 days; lanes 3 and 4, untreated/ddC-treated cells recovered for 2 days; and lanes 5 and 6, untreated/ddC-treated cells recovered for 5 days. (C) Schematic presentation of the two strand-specific probes (situated inside or outside the D-loop region) that were used for the experiment in (D). (D) Northern blot analysis of total RNA from ddC treated (+) or untreated (−) cells. Probes used are described in ‘Materials and Methods’ section and are depicted in (C). The levels of antisense transcript relative CYTB (%) are indicated. The experiments were repeated at least three times and one representative experiment is shown. The presented northern blots for the inside and outside probes represent two individual gels that have been run in parallel. For abbreviations, see legend to Figure 1.
Mentions: As shown in Figure 4A, three days of ddC treatment led to depletion of the full-length mtDNA (to about 20%). After the ddC had been removed from the media, five days of recovery were necessary to obtain a normal level of mtDNA. Southern blotting of untreated and ddC treated cells revealed that the ratio of 7S DNA to mtDNA was changed in the ddC treated cells compared to untreated controls. Three days of ddC treatment caused a strong reduction, but did not completely abolish 7S DNA levels (Figure 4B, lane 2). After removal of ddC, we saw a gradual recovery of 7S DNA levels relative full-length mtDNA (Figure 4B). This result is in agreement with the previous finding that the premature DNA replication termination event is less frequent after mtDNA depletion (50).

Bottom Line: The other motif, here denoted coreTAS, is at the D-loop 3'-end.Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication.Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, SE-405 30 Gothenburg, Sweden.

No MeSH data available.