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. 2018 Sep 20;46(20):10771–10781. doi: 10.1093/nar/gky852

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — The proportion of replication forks travelling towards, rather than away from, the unidirectional origin Ori-H is markedly increased in cells expressing transgenic wild-type Twinkle. Panels 1–4 Restriction digested HEK293T cell DNA separated by 1D-AGE and then subjected to an additional in-gel digestion step with another restriction enzyme, prior to standard 2D-AGE and gel processing. Fork direction is defined as clockwise (c), or anti-clockwise (ac) as indicated on the schematic map (bottom-right). Assignments of the arcs associated with fragment 1 of control cells (no transgene) and cells expressing wild-type Twinkle (Twk) are shown below the gel images; SC – strand coupled, SA – strand asynchronous replication. Twk and Twk-mut2 (D311A) transgenes were induced with 3 ng/mL doxycycline for 72 h. Immediately below each gel image (1–4) the relevant region of mtDNA is represented as a horizontal line, broken at the in-gel digestion site. Arrows above the line indicate the direction of fork travel, the thickness and length of the arrows correspond approximately to the proportion of forks travelling in a particular direction. Black arrows – forks travelling clockwise (towards Ori-H); gray arrows – forks travelling anti-clockwise (away from Ori-H). Black boxes indicate the position of the probes, h2, h4, h5 and h6.