Figure 3

Replication stress causes an excess of linear forms and results in topological modification of mtDNA. (A) Topology gel of undigested mtDNA from control and MGME1 knockout (KO) cells treated with ddC or UV; treatments as in Fig. 2. Under normal conditions, mtDNA in HEK293T cells exists as high molecular weight forms, including catenanes of two interlocked circles (cat), open circular monomers (oc) and supercoiled monomers (sc). Some mtDNA is linearized (lin) either naturally or due to extraction procedures. For the assignment of the different molecular forms see Pohjoismäki et al.³⁴ and Supplementary Fig. S4. While ddC or UV treatment do not have a major influence on the different forms in control cells, MGME1 knockout (KO) cells recovering from ddC exposure have novel molecular forms (black arrowheads) in addition to the 11-kb linear fragment (open arrowhead). (B) Long exposure of a separate re-run of the 72 h ddC samples. Note the excess smearing in the MGME1 knockout cells, probably due to mtDNA breakage. (C) 2D-AGE of undigested mtDNA from untreated and ddC-recovering parental and MGME1 knockout cells. The one-dimensional gel pattern from (A) is given below each panel to provide an approximate landmark. Notice the marked increase in linear forms in MGME1 knockout cells recovering from the ddC treatment. The additional bands marked with black arrowheads in (A) represent dimeric circular forms (2nc) and 33 kb dimeric linear mtDNA. An unusual eyebrow-arc (eb) is present in the MGME1 knockout cells, being more prominent in the cells recovering their mtDNA levels. (D) As the eb begins from the open circles and grows as an arc to the 2nc, it most likely represents replication intermediates broken at one end. (E) These broken intermediates are visible on 2D-AGE of mtDNA fragments cut once upstream of O_H, such as a PvuII digestion produces. Most replicating forms in PvuII -digested mtDNA should consist of replication bubbles (b), converting to double-y (dy) and termination (ter) intermediates close to the end of the molecule. If the replication bubble is broken at one end, the resulting intermediates will migrate on the y-arc (y). Note that some recently replicated circular forms are not cut by PvuII, because they either are partially single-stranded (ssc) or have RNA incorporation at the restriction site (oc). For further details regarding these forms and their connection to mtDNA replication, see Torregrosa-Muñumer et al.²². Again, the majority of the molecules are non-replicative and linearized (1n) by the restriction enzyme. (F) While the control cells have almost solely full-length replication bubbles, MGME1 knockout cells have mainly y-form replication intermediates, consistent with frequent breakage of the replication bubbles (see also Supplementary Fig. S5). The difference in the abundance of the circular forms (oc, ssc) between the wild-type and MGME1 knockout is likely caused by fewer molecules completing replication due to the premature arrest and breakage of the replication intermediates, resulting in depletion of the newly replicated molecules.