DEOXYNUCLEOTIDE POOL IMBALANCE, MITOCHONDRIAL DNA MAINTENANCE AND DISEASE

Nuclear DNA is synthesized only in growing cells and requires dNTPs produced by cytoplasmic "de novo" synthesis. Instead mt DNA replicates also in differentiated cells where de novo synthesis has stopped and dNTPs are produced inside mitochondria by enzymes encoded in nuclear genes. Mutations in some such genes are associated with depletion and mutations of mt DNA, probably via depletion of mt dNTP pools. Other disturbances of mt DNA maintenance are associated with genetic defects in enzymes that degrade dNTPs in the cytoplasm. It is believed that in these conditions mitochondria are damaged by the excess of dNTPs. The molecular mechanisms through which such enzyme deficiencies cause mt DNA instability were so far largely hypothetical due to lack of adequate methods to separately study mt and cytoplasmic dNTP pools. Within a research financed by Telethon, our group has devised and validated a new methodology to study the exchanges between mt and cytosolic dNTP pools. We will apply this method to investigate in cultured cells the effects of mutations in either of the two mt deoxynucleoside kinases, TK2 and dGK, and in two cytosolic enzymes involved in dNTP catabolism, thymidine phosphorylase (TP) and purine nucleoside phosphorylase (PNP). All these mutations cause severe human syndromes with a mt connection: mt DNA depletions (TK2 and dGK), MNGIE (TP) and T cell immunodeficiency (PNP). To separately inactivate each enzyme in cell cultures and mimick the effects of the gene mutations present in patients we will use a technique based on RNA interference. By the same means we will inactivate additional proteins involved in mt dNTP metabolism. Aim of our effort is that of obtaining a detailed picture of normal mt dNTP regulation and of how deficiency of individual enzymes can destabilize mt DNA and lead to mt disease. Our work may provide diagnostic methods to detect the molecular basis of still unexplained mt diseases and suggest possible treatment.