DNA precu[r]sor compartmentation in mammalian cells : metabolic and anti-metabolic studies of nuclear and mitochondrial DNA synthesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/8336h517d

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  • This dissertation describes an investigation of DNA precursor supply for mitochondrial and nuclear DNA synthesis in HeLa cells and Mouse L-cells. HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay for dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochondrial rNTP pools were also found to expand in response to these antimetabolites. The cellular dGTP pool is severely depleted by mtx and FUdR' which indicates dGTP levels might play an important role in mtx- and FUdR-induced cytotoxicity. Mouse L-cells were used to determine the relative contributions of an exogenously supplied precursor to nuclear and mitochondrial DNA replication. These experiments provide a dynamic representation of mitochondrial and nuclear DNA precusor metabolism. Cells were labeled to near steady state specific activities with ³²P-orthophosphate and subsequently labeled with [³H]uridine, a general pyrimidine precursor, in the continuing presence of ³²P. Cells were harvested at time intervals up to four days after [³H]uridine addition and mitochondrial and nuclear DNAs were purified. Deoxyribonucleoside monophosphates derived from these DNAs were separated by HPLC and the ³H/ ³²P ratio in each pyrimidine determined. The dCMP residues in mitochondrial DNA (mtDNA) were found to be derived exclusively from the exogenous supplied uridine. The dTMP residues from nuclear and mtDNA and the dCMP residues from nuclear DNA were seen to be synthesized partly from exogenous sources and partly from other sources, presumably de novo pyrimidine synthesis. The results suggest mtDNA replication can utilize exogenous dCTP precursors more efficiently than nuclear replication. Combined these results demonstrate that nuclear and mitochondrial DNA replication are supplied by distinct dNTP pools and regulation of dNTP biosynthesis is by different mechanisms in each of these two compartments.
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