Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells.

Carbonic anhydrase IX (CA IX) is highly expressed in rapidly proliferating and highly glycolytic cells, where it serves to enhance acid-regulatory capacity. Pulmonary microvascular endothelial cells (PMVECs) actively utilize aerobic glycolysis and acidify media, whereas pulmonary arterial endothelial cells (PAECs) primarily rely on oxidative phosphorylation and minimally change media pH. Therefore, we hypothesized that CA IX is critical to PMVEC angiogenesis because of its important role in regulating pH. To test this hypothesis, PMVECs and PAECs were isolated from Sprague-Dawley rats. CA IX knockout PMVECs were generated using the CRISPR-Cas9 technique. During serum-stimulated growth, mild acidosis (pH 6.8) did not affect cell counts of PMVECs, but it decreased PAEC cell number. Severe acidosis (pH 6.2) decreased cell counts of PMVECs and elicited an even more pronounced reduction of PAECs. PMVECs had a higher CA IX expression compared with PAECs. CA activity was higher in PMVECs compared with PAECs, and enzyme activity was dependent on the type IX isoform. Pharmacological inhibition and genetic ablation of CA IX caused profound dysregulation of extra- and intracellular pH in PMVECs. Matrigel assays revealed impaired angiogenesis of CA IX knockout PMVECs in acidosis. Lastly, pharmacological CA IX inhibition caused profound cell death in PMVECs, whereas genetic CA IX ablation had little effect on PMVEC cell death in acidosis. Thus CA IX controls PMVEC pH necessary for angiogenesis during acidosis. CA IX may contribute to lung vascular repair during acute lung injury that is accompanied by acidosis within the microenvironment.

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.