Fig 2: Replication stress causes loss of nascent mtDNA in FANCO/RAD51C patient cells.(A) Representative images of MIRA with EdU label incorporated immediately before treatment with mtOX (4 µM), aphidicolin (APH; 7 µM), or no treatment. (B) Scatterplot quantification of (A). Top: Experimental sketch. (C) Scatterplot quantification of MIRA signals in S-phase cells and non–S phase in SH2038 cells following the experimental schemes in (B). (D) Scatterplot quantification of MIRA with or without treatment with MitoPQ (10 µM) for 3 hours after EdU incubation. Top: Experimental sketch. (E) mtDNA copy numbers measured by quantitative polymerase chain reaction (qPCR) without treatment, with mtOX, or with aphidicolin. (F) Scatter dot plot quantification MIRA with EdU labeled incorporated 3 hours after treatment with mtOX or aphidicolin, respectively. Top: Experimental sketch. Bars represent the mean of compiled data from biological repeats. P values for scatter dot blots are derived using the Mann-Whitney test and, for bar graph (mtDNA content), by Student’s t test. n.s., not significant; ****P < 0.0001, ***P < 0.001, and **P < 0.01.

Fig 3: Mitochondria remain normal with replication fork degradation.(A) Western blot of mitochondrial respiratory proteins and autophagy marker LC3 with or without mtOX for 3 or 24 hours and MRE11 inhibition by mirin. (B) Electron microscope images of SH2038 and RAD51C+ cells with and without mtOX treatment. (C) Bar graph quantification of mitochondrial membrane potential as measured by MitoTracker CMXRos 3 hours after treatment with mtOX (4 µM) or carbonyl cyanide m-chlorophenyl hydrazine (CCCP), used as a positive control. Error bars represent SD. a.u., arbitrary units. (D) Clonogenic survival of SH2038 FANCO patient cells and RAD51C+ cells with and without mtDNA replication stalling by mtOX, normalized to RAD51C+ without treatment. Error bars represent SEM. (E) Representative images of (C). Bars represent the mean of compiled data from biological repeats, and P values are derived using Student’s t test. ****P < 0.0001, ***P < 0.001, and **P < 0.01.

Fig 4: MIRA detects nascent mtDNA.(A) Schematic of MIRA. Incorporated EdU (green) is detected by PLA amplification (pink circles). Ab, antibody. (B) Representative image of wild-type RAD51C-complemented FA patient cells (RAD51C+) cells with antibody stain against mitochondria (mt-protein) and mito-FISH probes. (C) Representative images of MIRA signals in RAD51C+ cells with antibody stain against mitochondria (mt-protein). (D) Airyscan superresolution image of MIRA signals in RAD51C+ cells with antibody stain against double-stranded DNA (dsDNA). (E) Quantification of (D) MIRA signals overlapping (black bar) or not overlapping (gray bar) with dsDNA. (F) Airyscan superresolution image of MIRA signals in RAD51C+ cells with antibody stain against TFAM. (G) Quantification of (F) MIRA signals overlapping (black) or not (gray) with TFAM. (H) Representative images of MIRA in RAD51C+ cells with and without ethidium bromide (EtBr; 50 ng/ml) before and during EdU labeling. NT, no treatment. (I) Quantification of (H). Top: Experimental scheme. (J) Representative images of MIRA (red) in rho zero SH2038 cells with PicoGreen DNA stain (green). (K) Quantification of (J). (L) Quantification of MIRA signals in SH2038 S-phase cells (green) and non–S-phase cells (gray). Bars represent the mean of compiled data from biological repeats. P values are derived using the Mann-Whitney test. ****P < 0.0001.

Fig 5: MRE11 nuclease causes mtDNA degradation in FANCO/RAD51C patient cells.(A) Representative images of MIRA with and without with mtOX (4 µM) and chemical inhibition of MRE11 nuclease with mirin (50 µM). Top: Experimental sketch. (B) Scatter dot plot quantification of (A), MIRA signals. (C) Scatter dot plot quantification of MIRA with and without mtOX and inhibition of MRE11 nuclease with PFM39 (100 µM), following the experimental schemes in (B). (D) Representative images of a MIRA with and without mitochondrial replication stalling with ddC (20 µM) in the presence or absence of mirin (50 µM). Top: Experimental sketch. (E) Scatter dot plot quantification of (D). (F) Scatter dot plot quantification of MIRA signals in SH2038 cells with MRE11 small interfering RNA (siRNA) knockdown (siMRE11) or control siRNA (siCtrl). Top: Experimental sketch. Inset: Western blot analysis of the MRE11 knockdown. GAPDH, glyceraldehyde phosphate dehydrogenase. (G) Representative images of a MIRA in BJ fibroblast with siRNA knockdown of RAD51C (siRAD51C) or siCtrl. Top: Experimental sketch. (H) Scatter dot plot quantification of (G). Inset: Western blot analysis of the RAD51C knockdown. Bars represent the mean of compiled data from biological repeats. P values are derived using the Mann-Whitney test. ****P < 0.0001, ***P < 0.001, and *P < 0.05.