Ribonucleotide reductase association with mammalian liver mitochondria.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Mitochondrial deoxyribonucleoside triphosphate (dNTP) pools are highly asymmetric.
• This asymmetry is linked to the elevated mutation rate of the mitochondrial genome compared to the nuclear genome.
• Understanding dNTP synthesis and accumulation pathways within mitochondria is crucial.

Purpose of the Study:

• To identify pathways for dNTP synthesis and accumulation within mammalian mitochondria.
• To investigate the presence and activity of ribonucleotide reductase in mitochondria.

Main Methods:

• Enzymatic assays to detect ribonucleotide reductase activity in isolated mitochondria.
• Mass spectrometry to identify proteins in purified mitochondria, specifically immunoprecipitated proteins.
• Incubation of respiring mitochondria with radiolabeled nucleotide diphosphates to track dNTP accumulation.

Main Results:

• Ribonucleotide reductase activity was identified specifically associated with mammalian tissue mitochondria.
• Mass spectrometry revealed the R1 subunit of ribonucleotide reductase in purified mitochondria.
• Enzymatic and immunological activity of ribonucleotide reductase was detected in rat liver mitochondrial nucleoids.
• Incubation with radiolabeled cytidine diphosphate and guanosine diphosphate resulted in the accumulation of corresponding radiolabeled dNTPs within mitochondria.

Conclusions:

• Mammalian mitochondria possess the machinery for ribonucleotide reduction, indicating potential for internal dNTP synthesis.
• Mitochondrial ribonucleotide reduction is a significant contributor to mitochondrial dNTP pools.
• This internal synthesis pathway may explain the observed asymmetry in mitochondrial dNTP pools and contribute to the mitochondrial mutation rate.