Pathology of dNTP dysregulation

• Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac abnormalities and impaired cardiac conduction systems. As a result, they died between two and four weeks after birth. Taken together, our studies provide the first empirical evidence that both the de novo and salvage pathways are essential to keeping the dNTP concentration at an optimal range to prevent mutagenesis, carcinogenesis, and mortality.

Nyckelord

dNTP metabolism

DNA replication

mutation rate

cancer

heart development

RNR

SAMHD1

Rrm1

Samhd1

medicinsk biokemi

Medical Biochemistry

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