Abstract
Defects in nucleotide metabolism and imbalances in deoxynucleotide triphosphate (dNTP) pools are associated with several human diseases, including cancer and mitochondrial disorders. In non-replicative cells, while DNA synthesis is reduced, a continuous supply of nucleotides is essential to sustain mitochondrial DNA (mtDNA) replication and repair. Human all-α dCTP pyrophosphatase 1 (DCTPP1), a nucleotido hydrolase with high specificity for dCTP, plays a critical role in maintaining nucleotide homeostasis, however its participation in mtDNA stability remains unexplored. In this study we performed a detailed analysis of pyrimidine metabolism enzymes in non-dividing cells. We found that during quiescence, DCTPP1 is predominantly localized to mitochondria. Depletion of the enzyme leads to upregulation of the de novo thymidylate synthesis pathway and expansion of both the dCTP and dGTP pools, highlighting its pivotal role in regulating the dNTP balance. To explore the potential therapeutic relevance of these observations, we used an in vitro model of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare mitochondrial disorder caused by thymidine phosphorylase (TP) deficiency and characterized by dCTP depletion and mtDNA loss. Long-term thymidine overloading in quiescent cells (a model mimicking TP deficiency) led to reduced dCTP levels and the depletion of mtDNA, effects that were reversed upon DCTPP1 knockdown. Hence, reduced DCTPP1 levels restored dCTP availability and increased mtDNA copy number. These findings suggest that DCTPP1 plays a critical role in regulating mitochondrial dNTP pools and that down-regulation of the enzyme may serve as a compensatory mechanism in disorders marked by secondary dCTP depletion. DCTPP1 may therefore represent a promising therapeutic target for mitochondrial DNA depletion syndromes such as MNGIE.
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Acknowledgements
We thank the IPBLN core facility, especially Laura Montosa for her support with advanced imaging resources and microscopy analysis, and Aurora Constán for her technical assistance. This work was funded by a grant from the Junta de Andalucía (P18 -RT-4870) and by the grant PID2022-142971OB-I00 MCIN/AEI/ https://doi.org/10.13039/501100011033 / ERDF, UE.
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DGP and LMRP contributed to the conceptualization, resources, data curation, supervision, and funding acquisition. BF also contributed to the conceptualization, methodology, and validation of the investigation. BF, GPM, BMA, AEV, and DGP designed the experiments, while BF prepared all the figures. DGP and BF were responsible for writing the original draft and for the writing – review and editing. AEV, GPM, BMA and LMPR participated in the revision of the manuscript.
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Fernández, B., Pérez-Moreno, G., Martínez-Arribas, B. et al. DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells. Cell Death Dis (2026). https://doi.org/10.1038/s41419-026-08632-1
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DOI: https://doi.org/10.1038/s41419-026-08632-1
