Graduate Thesis Or Dissertation
 

Insights into mammalian DNA replication from analysis of mutations affecting deoxyribonucleotide biosynthesis

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  • Although the synthesis of DNA precursors is closely coordinated with DNA replication, it is still not clear whether deoxyribonucleoside triphosphates (dNTPs) influence DNA replication independently of their interactions with DNA polymerase catalytic sites. In an effort to understand the extent to which rate and fidelity of DNA replication are regulated by its precursors, two distinct mammalian cell mutants, which are defective in deoxyribonucleotide biosynthesis, have been analyzed. JB3-B is a Chinese hamster ovary (CHO) cell mutant that is temperature-sensitive for DNA replication. Measurement of dNTP pools as a function of time after shift of cultures to the non-permissive temperature revealed that all four dNTP pools declined at similar rates in extracts of both rapidly isolated nuclei and whole cells. The magnitude and time course of the pool size changes measured in nuclear extracts more closely corresponded to the inhibition of DNA synthesis than did those measured in whole-cell extracts. Ribonucleotide reductase activity was diminished in extracts prepared at the non-permissive temperature, in parallel with the decline in dNTP pool sizes. Moreover, the activity of a cell extract was thermolabile in vitro. The data suggest that the intranuclear concentrations of the four dNTPs determine the rate of DNA replication in JB3-B under the conditions of limited ribonucleotide reductase activity. Thy- 49 and Thy- 303 are CHO cell mutants that have a mutator phenotype, but they differ in the extents to which spontaneous mutagenesis is stimulated. Both strains have imbalanced dNTP pools that result from loss of allosteric regulation of CTP synthetase. When the DNA precursor pools from S phase-enriched cells were analyzed, large imbalances were seen in the extracts of rapidly isolated nuclei, while whole-cell extracts showed smaller biases, in both strains. The specific nuclear dCTP pool increase produced the large perturbations in the balance of nuclear precursor pools. Thy- 303, which has higher mutation frequencies than Thy- 49, showed the more aberrant precursor pools. The nuclear pool size changes were correlated with the frequencies of mutations observed in the two strains. The data suggest that the intranuclear balance of DNA precursor pools determines the fidelity of DNA replication.
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