| Summary: | It is well known that the mitochondrial genome has a much higher spontaneous
mutation rate than the nuclear genome. mtDNA mutations have been identified in
association with many diseases and aging. mtDNA replication continues throughout the
cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is
required for replication and maintenance of the mitochondrial genome. However, it is not
clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are
regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and
nucleotide metabolism is associated with several human diseases. Clearly, to uncover the
pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis,
information is needed regarding dNTP biosynthesis and maintenance within
mitochondria, and biochemical consequences of disordered mitochondrial dNTP
metabolism.
The studies described in this thesis provide important insight into these questions.
First, we found that a distinctive form of ribonucleotide reductase is associated with
mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP
synthesis within mitochondria. Second, we found that long term thymidine treatment
could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from
thymidine treatment could account for the spectrum of mtDNA point mutations found in
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results
support the proposed pathogenesis of this disease. Third, we found that normal
intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity
studies show that these imbalanced pools can stimulate base substitution and frameshift
mutations, with a substitution pattern that correlates with mitochondrial substitution
mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool
asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases.
Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by
insufficient transport of dNTPs into mitochondria resulting from a loss-of-function
mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found
that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA
patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous
mutant cells extracted from Dnc gene knockout mouse embryos. These results do not
support the proposed pathogenesis of this disease and indicate that the DNC protein does
not play a crucial role in the maintenance of intramitochondrial dNTP pools. === Graduation date: 2005
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