Figure 4
mtDNA copy number recovery is impaired by the loss of MGME1. (A) 72 h ddC treatment of HEK293T, MGME1 overexpressor and MGME1 knockout cells, followed by recovery for 48 and 96 h. As evident from the severe copy number depletion after 72 h, ddC treatment results in an almost complete block of mtDNA synthesis. Parental HEK293T cells recover their mtDNA copy number to normal levels within 96 h after the removal of the drug, whereas the recovery is severely impaired in the MGME1 knockout (KO) cells. All samples are normalized to the untreated HEK293T mtDNA copy number (100%) to show the differences in basal mtDNA levels. Results are presented as mean ± SD (n = 3, from at least two independent experiments) and p values based on one-way ANOVA with post-hoc Tukey HSD Test. (B) 2D-AGE analysis of the DraI 12,273–16,012 fragment from ddC treated (200 µM ddC for 72 h) parental 293 T-REx cells (control) and MGME1 T-REx cells induced with 5 ng/ml doxycycline. (C) Identical analysis for regular HEK293 cells (control) and MGME1 knockout (KO) cells. Treatment with S1 nuclease (right panels) reveals the fully double-stranded intermediates. The lower panels represent longer exposures of the upper ones and the exposures between adjacent panels are comparative. The treatment stalls all mtDNA replication intermediates and induces replication initiation outside of OH (b, see Fig. 1) in all cells except MGME1 KO. Regressed forks (rx´) appear different in the ddC treated cells compared to the UV (rx in Figs 1 and 2) and merge with the x-arc. Note the lack of a bubble arc, the prominent x-forms and an unusual arc (arrow) arising from x´s in the MGME1 knockout cells.
