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Mitochondrial diseases are estimated to affect one child out of every 5,000 born worldwide. A major subgroup, mitochondrial DNA depletion syndromes (MDDS), is defined by failure to maintain optimal mitochondrial DNA (mtDNA) copy number. Severe MDDS causes progressive mitochondrial dysfunction and early mortality, but treatment options are limited by knowledge gaps in the interplay between the metabolic pathways that supply DNA building blocks (dNTPs). An MDDS variant arises from deficiency in RRM2B, a protein required for dNTP synthesis in non-dividing cells. To determine how MDDS-associated pathologies could be mitigated, my research analysed RRM2B-deficient mice as an MDDS model.

This thesis describes how the balance between RRM2B-dependent synthesis and SAMHD1-mediated degradation controls dNTP availability in non-dividing tissues, establishes purine-selective dNTP depletion as a central biochemical feature of RRM2B-associated MDDS, and explains differences in disease onset and tissue vulnerability. We achieved these goals by first evaluating how the loss of RRM2B impacts dNTP concentrations and mtDNA copy number, as well as mouse physiology and survival. We observed that RRM2B deficiency shortened mouse lifespan due to depletion of purine dNTPs and mtDNA copy number, along with structural deterioration of specific tissues, detectable only after birth. We then determined how these parameters changed with concurrent deletion of SAMHD1, a major dNTP-degrading protein, as a countermeasure.

Deletion of SAMHD1 countered the MDDS phenotype by elevating purine dNTP concentrations and mtDNA copy number. This extended mouse survival significantly, with a clear regression of structural deformities in key tissues such as the kidneys and skeletal muscle. Interestingly, the extent of dNTP recovery was tissue-dependent, being highest in the liver and spleen, possibly due to the activity of other dNTP-producing enzymes, and/or differential SAMHD1 activity in these tissues.

Importantly, the partial rescue achieved by deleting SAMHD1 provides a mechanistic rationale for a therapeutic strategy that combines increasing dNTP supply with inhibiting dNTP degradation.