Live-cell imaging following UVC treatment indicates increased mtDNA degradation.

A) Representative images for control (left) and UVC treated (right) cells. Merged channels include mitotracker (red, stains mitochondria), lysotracker (pink, stains lysosomes), picogreen (green, stains mtDNA), and nuclei (blue). B) Inset outlined in the UVC treated cells in panel A. Channels from left to right include mitochondria, mtDNA, lysosome, and the merged image. The yellow arrow indicates a mtDNA spot colocalized with lysotracker. For panels C-F, x-axes represent the treatment and data was analyzed via two-tailed unpaired t-test. Data includes at least n=30 cells per treatment group per imaging session and three distinct imaging sessions. C) Quantification of the total mitochondrial area per cell normalized to the size of the cell (p<0.0001). D) Quantification of the number of lysosomes normalized to the size of the cell (p=0.0054). E) Quantification of the number of mtDNA spots normalized to the size of the cell (p<0.0001). F) Quantification of the proportion of mtDNA spots within lysosomes (p<0.0001).

UVC exposures stimulate mtDNA turnover to remove mtDNA damage, upregulation in mtDNA replication genes, and increase mitochondrial transcription in the absence of apparent mitochondrial dysfunction.

For panels A-F, x-axes represent recovery time, i.e., time following the UVC exposure. Doses of UVC used were 0, 10, and 30 J/m2. A) Mitochondrial DNA damage levels following UVC exposure. Y-axis represents the level of damage (lesions/10kb). Data analyzed via two-way ANOVA with a Dunnett’s post-hoc test for multiple comparisons (dose: p<0.0001, time: p=0.0001, interaction: p=0.0024). For panels B-E, y-axes represent fold change normalized to the control (0 J/m2) at each timepoint. All data was analyzed via two-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. B) TFAM expression level assessed via qPCR following UVC exposure (dose: p=0.004, time: p=0.001, interaction: p=0.02). C) POLG expression level assessed via qPCR following UVC exposure (dose: p=0.002, time: p=0.003, interaction: p=0.004). D) POLRMT expression level assessed via qPCR following UVC exposure (Dose: p=0.015, time: p=0.009, interaction: p=0.12). E) ND-1 expression level assessed via qPCR following UVC exposure (Dose: p<0.0001, time: p=0.006, interaction: p=0.003). F) Mitochondrial membrane potential assessed via flow cytometry following exposure to 0, 10, 30, or 50 J/m2 UVC at 6 and 24 hours after exposure. The x-axis represents the exposure group, and the y-axis represents the change in Median Fluorescent Intensities (MFI) of TMRM normalized to the control for each timepoint. Cells were also exposed to FCCP, a well-known chemical that causes mitochondrial depolarization, as a positive control. Data was analyzed via a two-way ANOVA (treatment: p=0.0008, time: p<0.0001, interaction: p=0.14).

TFAM has specific binding across the mitochondrial genome and exhibits a reduction in specificity in the context of UVC-irradiated DNA.

The median z-score plotted to the coordinate of the middle nucleotide of the variable mitochondrial region of the sequence for the non-UVC-irradiated chamber containing 30 nM TFAM (A) and the UVC-irradiated chamber containing 30 nM TFAM (B). The gene map of the mitochondrial genome is shown in the center. Z-score variation is color coded such that positive z-scores associated with high binding are in in blue, and progressively get lighter as the z-scores get higher. Negative z-scores associated with low binding are in red. Regions highlighted in yellow are the promoter sequences of the mitochondrial genome on the light strand (LSP1 and LSP2), while regions highlighted in pink are the promoter sequences on the heavy strand (HSP1 and HSP2). Plots of these regions can be found in Figure S12.

TFAM KD measurements:

Atomic force microscopy of TFAM-DNA substrates indicates an increase in compaction associated with UVC exposure.

A) AFM images of the pUC19 DNA only (control) and the 30 nM TFAM-DNA complexes in two different conditions: one with (+UV) or without (-UV) UVC irradiated DNA. Colored arrows on the AFM images represent the different TFAM-DNA complexes categorizations: dispersed (green), intermediate (yellow), punctate (red), and free DNA (blue). The white scale bar represents 1 μm. B) Histogram plot of the volumes distribution of plasmid DNA only (control) as well as the 15 nM and 30 nM TFAM-DNA complexes with (+UV) or without (-UV) UVC damaged DNA. All axes in the histogram are scaled the same. The data for control pUC19 only was replicated for each TFAM concentration for clarity in comparisons. C) Atomic force microscopy images of three different TFAM-DNA complex categorizations labeled as dispersed (small clusters with no protein tracts on DNA), intermediate (small clusters with protein tracts), and punctate (tightly associated punctate clusters). The white scale bar represents 200 nm. D) A bar graph representing the percent total number of plasmids in the 15nM TFAM concentration with (+UV) (N=66) or without (-UV) (N=136) UVC damaged DNA and the 30nM TFAM concentration with (+UV) (N=65) or without (-UV) (N=91) UVC damaged DNA. Detailed counts of each classification can be found in table S1. E) Schematic of the TFAM-DNA binding and compaction mechanism.

Increased nucleoid compaction does not protect mtDNA from UVC-induced DNA damage or alter damage removal rates.

A) Exposure paradigm for TFAM overexpression experiments. Cells were exposed to doxycycline for 48 hours prior to UVC exposure to ensure upregulation of TFAM at the time of exposure. Protein was quantified in control cells only to ensure TFAM upregulation. B) Representative western blot of TFAM-tetON cell lysates following 48, 72, and 96 hours of doxycycline treatment to confirm TFAM upregulation. TFAM-tetON cells contain an HA tag that when expressed, results in a second band. C) Pixel quantifications of n=3 western blots shown in panel (B) at each time point. TFAM protein levels were normalized to β-actin and then normalized to the non-doxycycline treated controls. X-axis represents the doxycycline treatment and y-axis represents the fold change relative to the non-doxycycline treated cells. Data was analyzed via two-way ANOVA (doxycycline treatment p=0.0006, time p=0.25, interaction p=0.25). D) TFAM mRNA quantification following doxycycline treatment. X-axis represents the doxycycline treatment and y-axis represents the fold change relative to the non-doxycycline treated cells. Data was analyzed via two-way ANOVA (doxycycline treatment p=0.0003, time p=0.52, interaction p=0.52). E) Lesion frequency following UVC exposure in TFAM-tetON cells immediately after the exposure (0-hour recovery time). X-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10kb). Data was analyzed via two-way ANOVA (UVC dose: p<0.0001, doxycycline treatment p=0.15, interaction: p=0.77). F) Lesion frequency following UVC exposure in TFAM-tetON cells 24 hours after the exposure (24-hour recovery time). X-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10kb). Data was analyzed via two-way ANOVA (UVC dose: p<0.0001, doxycycline treatment p=0.71, interaction: p=0.65). G) Lesion frequency following UVC exposure in TFAM-tetON cells 48 hours after the exposure (48-hour recovery time). X-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10kb). Data was analyzed via two-way ANOVA (UVC dose: p<0.0001, doxycycline treatment p=0.01, interaction: p=0.61). H) Representative AFM images of in vitro nucleoids generated using purified TFAM and PCR amplified human mtDNA at 0, 100, 250, and 1000 nM TFAM. All scale bars represent 500 nm. I) Lesion frequency following UVC exposure in in vitro nucleoids. X-axis represents the UVC dose and y-axis represents the lesion frequency (lesions per 10kb). Data was analyzed via two-way ANOVA (UVC dose: p<0.0001, TFAM concentration p=0.20, interaction: p=0.86).

Proposed cellular model of the role of TFAM in mtDNA damage sensing.

Alterations in TFAM-DNA interactions may alter compactional status of mitochondrial nucleoids. This feature may serve to “tag” mitochondrial genomes as damaged, which could lend itself to repression of the replication of damaged genomes, flagging damaged genomes for targeted degradation, or both. Both active removal and repression of replication would allow for removal of damaged genomes during mtDNA turnover processes and provide a mechanism for preventing mtDNA mutagenesis. Created with BioRender.com.