DEOXYNUCLEOTIDE POOL IMBALANCE, MITOCHONDRIAL DNA MAINTENANCE AND DISEASE

DNA synthesis requires 4 deoxynucleotides (dNTPs) that are made by 2 pathways, de novo synthesis in the cytosol and salvage, both in the cytosol and in mitochondria. De novo synthesis was believed to be limited to dividing cells. It has now been recognized that it occurs also in quiescent cells and that mutations in the responsible enzyme cause lethat mitochondrial (mt) DNA depletions. Also mutations in mt enzymes involved in the salvage pathway cause mtDNA depletion and lead to death in children. Thus both pathways are required to supply building blocks for mtDNA in non dividing cells. The enzymes of the 2 pathways create in the cells a functional network that includes also enzymes that degrade deoxynucleotides and thereby maintain the correct balance among the 4 dNTPs. Also mutations in degradative enzymes destabilize mtDNA, because they cause imbalance of the dNTP pools. All these enzymes are encoded in nuclear genes and in the patients the mutations are present in all cells of the body. However, only selected organs display the mtDNA depletion. We study how the enzyme network regulating the balance of dNTP pools in mitochondria is organized and functions. In previous work supported by Telethon we have devised a new method to study dNTP pools in mitochondria and discovered that cytosolic de novo synthesis supports mt pools in both dividing and resting cells and it can compensate defects in the mt salvage pathways. Now we want to understand why some specific organs are preferential targets of mutations in specific enzymes of the network. To do this we first try to identify all the players in the regulation of mt dNTP pools in fibroblasts. To recognize the proteins involved we reduce their levels by chemical inhibition, mutation, or RNA-interference. Then we compare the picture obtained in fibroblasts with that of cultured muscle cells. Differences in the organization of the network may become apparent and suggest approaches to compensate the enzyme deficiencies.