UV irradiation alters TFAM binding to mitochondrial DNA

Reviewer #1 (Public review):

Summary:

The authors investigate how UVC-induced DNA damage alters the interaction between the mitochondrial transcription factor TFAM and mtDNA. Using live-cell imaging, qPCR, atomic force microscopy (AFM), fluorescence anisotropy, and high-throughput DNA-chip assays, they show that UVC irradiation reduces TFAM sequence specificity and increases mtDNA compaction without protecting mtDNA from lesion formation. From these findings, the authors suggest that TFAM acts as a "sensor" of damage rather than a protective or repair-promoting factor.

Strengths:

(1) The focus on UVC damage offers a clean system to study mtDNA damage sensing independently of more commonly studied repair pathways, such as oxidative DNA damage. The impact of UVC damage is not well understood in the mitochondria, and this study fills that gap in knowledge.

(2) In particular, the custom mitochondrial genome DNA chip provides high-resolution mapping of TFAM binding and reveals a global loss of sequence specificity following UVC exposure.

(3) The combination of in vitro TFAM DNA biophysical approaches, combined with cellular responses (gene expression, mtDNA turnover), provides a coherent multi-scale view.

(4) The authors demonstrate that TFAM-induced compaction does not protect mtDNA from UVC lesions, an important contribution given assumptions about TFAM providing protection.

Weaknesses:

(1) The authors show a decrease in mtDNA levels and increased lysosomal colocalization but do not define the pathway responsible for degradation. Distinguishing between replication dilution, mitophagy, or targeted degradation would strengthen the interpretation

(2) The sudden induction of mtDNA replication genes and transcription at 24 h suggests that intermediate timepoints (e.g., 12 hours) could clarify the kinetics of the response and avoid the impression that the sampling coincidentally captured the peak.

(3) The authors report no loss of mitochondrial membrane potential, but this single measure is limited. Complementary assays such as Seahorse analysis, ATP quantification, or reactive oxygen species measurement could more fully assess functional integrity.

(4) The manuscript briefly notes enrichment of TFAM at certain regions of the mitochondrial genome but provides little interpretation of why these regions are favored. Discussion of whether high-occupancy sites correspond to regulatory or structural elements would add valuable context.

(5) It remains unclear whether the altered DNA topology promotes TFAM compaction or vice versa. Addressing this directionality, perhaps by including UVC-only controls for plasmid conformation, would help disentangle these effects if UVC is causing compaction alone.

(6) The authors provide a discrepancy between the anisotropy and binding array results. The reason for this is not clear, and one wonders if an orthogonal approach for the binding experiments would elucidate this difference (minor point).

Assessment of conclusions:

The manuscript successfully meets its primary goal of testing whether TFAM protects mtDNA from UVC damage and the impact this has on the mtDNA. While their data points to an intriguing model that TFAM acts as a sensor of damaged mtDNA, the validation of this model requires further investigation to make the model more convincing. This is likely warranted for a follow-up study. Also, the biological impact of this compaction, such as altering transcription levels, is not clear in this study.

Impact and utility of the methods:

This work advances our understanding of how mitochondria manage UVC genome damage and proposes a structural mechanism for damage "sensing" independent of canonical repair. The methodology, including the custom TFAM DNA chip, will be broadly useful to the scientific community.

Context:

The study supports a model in which mitochondrial genome integrity is maintained not only by repair factors, but also by selective sequestration or removal of damaged genomes. The demonstration that TFAM compaction correlates with damage rather than protection reframes an interesting role in mtDNA quality control.

Reviewer #2 (Public review):

Summary:

King et al. present several sets of experiments aimed to address the potential impact of UV irradiation on human mitochondrial DNA as well as the possible role of mitochondrial TFAM protein in handling UV-irradiated mitochondrial genomes. The carefully worded conclusion derived from the results of experiments performed with human HeLa cells, in vitro small plasmid DNA, with PCR-generated human mitochondrial DNA, and with UV-irradiated small oligonucleotides is presented in the title of the manuscript: "UV irradiation alters TFAM binding to mitochondrial DNA". The authors also interpret results of somewhat unconnected experimental approaches to speculate that "TFAM is a potential DNA damage sensing protein in that it promotes UVC-dependent conformational changes in the [mitochondrial] nucleoids, making them more compact." They further propose that such a proposed compaction triggers the removal of UV-damaged mitochondrial genomes as well as facilitates replication of undamaged mitochondrial genomes.

Strengths:

(1) The authors presented convincing evidence that a very high dose (1500 J/m2) of UVC applied to oligonucleotides covering the entire mitochondrial DNA genome alleviates sequence specificity of TFAM binding (Figure 3). This high dose was sufficient to cause UV lesions in a large fraction of individual oligonucleotides. The method was developed in the lab of one of the corresponding authors (reference 74) and is technically well-refined. This result can be published as is or in combination with other data.

(2) The manuscript also presents AFM evidence (Figure 4) that TFAM, which was long known to facilitate compaction of the mitochondrial genome (Alam et al., 2003; PMID 12626705 and follow-up citations), causes in vitro compaction of a small pUC19 plasmid and that approximately 3 UVC lesions per plasmid molecule result in a slight, albeit detectable, increase in TFAM compaction of the plasmid. Both results can be discussed in line with a possible extrapolation to in vivo phenomena, but such a discussion should include a clear statement that no in vivo support was provided within the set of experiments presented in the manuscript.

Weaknesses:

Besides the experiments presented in Figures 3 and 4, other results do not either support or contradict the speculation that TFAM can play a protective role, eliminating mitochondrial genomes with bulky lesions by way of excessive compaction and removing damaged genomes from the in vivo pool.

To specify these weaknesses:

(1) Figure 1 - presents evidence that UVC causes a reduction in the number of mitochondrial spots in cells. The role of TFAM is not assessed.

(2) Figure 2 - presents evidence that UVC causes lesions in mitochondrial genomes in vivo, detectable by qPCR. No direct assessment of TFAM roles in damage repair or mitochondrial DNA turnover is assessed despite the statements in the title of Figure 2 or in associated text. Approximately 2-fold change in gene expression of TFAM and of the three other genes does not provide any reasonable support to suggestion about increased mitochondrial DNA turnover over multiple explanations on related to mitochondrial DNA maintenance.

(3) Figure 5. Shows that TFAM does not protect either mitochondrial nucleoids formed in vitro or mitochondrial DNA in vivo from UVC lesions as well as has no effect on in vivo repair of UV lesions.

(4) Figure 6: Based on the above analysis, the model of the role of TFAM in sensing mtDNA damage and elimination of damaged genomes in vivo appears unsupported.

(5) Additional concern about Figure 3 and relevant discussion: It is not clear if more uniform TFAM binding to UV irradiated oligonucleotides with varying sequence as compared to non-irradiated oligonucleotides can be explained by just overall reduced binding eliminating sequence specific peaks.

Reviewer #3 (Public review):

Summary:

The study is grounded in the observations that mitochondrial DNA (mtDNA) exhibits a degree of resistance to mutagenesis under genotoxic stress. The manuscript focuses on the effects of UVC-induced DNA damage on TFAM-DNA binding in vitro and in cells. The authors demonstrate increased TFAM-DNA compaction following UVC irradiation in vitro based on high-throughput protein-DNA binding and atomic force microscopy (AFM) experiments. They did not observe a similar trend in fluorescence polarization assays. In cells, the authors found that UVC exposure upregulated TFAM, POLG, and POLRMT mRNA levels without affecting the mitochondrial membrane potential. Overexpressing TFAM in cells or varying TFAM concentration in reconstituted nucleoids did not alter the accumulation or disappearance of mtDNA damage. Based on their data, the authors proposed a plausible model that, following UVC-induced DNA damage, TFAM facilitates nucleoid compaction, which may serve to signal damage in the mitochondrial genome.

Strengths:

The presented data are solid, technically rigorous, and consistent with established literature findings. The experiments are well-executed, providing reliable evidence on the change of TFAM-DNA interactions following UVC irradiation. The proposed model may inspire future follow-up studies to further study the role of TFAM in sensing UVC-induced damage.

Weaknesses:

The manuscript could be further improved by refining specific interpretations and ensuring terminology aligns precisely with the data presented.

(1) In line 322, the claim of increased "nucleoid compaction" in cells should be removed, as there is a lack of direct cellular evidence. Given that non-DNA-bound TFAM is subject to protease digestion, it is uncertain to what extent the overexpressed TFAM actually integrates into and compacts mitochondrial nucleoids in the absence of supporting immunofluorescence data.

(2) In lines 405 and 406, the authors should avoid equating TFAM overexpression with compaction in the cellular context unless the compaction is directly visualized or measured.

(3) In lines 304 and 305 (and several other places throughout the manuscript), the authors use the term "removal rates". A "removal rate" requires a direct comparison of accumulated lesion levels over a time course under different conditions. Given the complexity of UV-induced DNA damage-which involves both damage formation and potential removal via multiple pathways-a more accurate term that reflects the net result of these opposing processes is "accumulated DNA damage levels." This terminology better reflects the final state measured and avoids implying a single, active 'removal' pathway without sufficient kinetic data.

(4) In line 357, the authors refer to the decrease in the total DNA damage level as "The removal of damaged mtDNA". The decrease may be simply due to the turnover and resynthesis of non-damaged mtDNA molecules. The term "removal" may mislead the casual reader into interpreting the effect as an active repair/removal process.