Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not
symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in
cells. In an in vitro replication system involving semiconservative replication from
an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human
cell extracts in reaction mixtures containing "biologically biased" dNTP pools
estimated from HeLa cell nuclei is not significantly different from that seen when
replication is done with equimolar dNTP concentrations. Significant reduction of
dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations
during replication reaction also did not increase mutation frequency. In contrast, in
vitro replication with dNTP concentrations calculated from normal diploid fibroblast
cells, which are three- to four-fold lower in concentrations, showed a marked
reduction of the observed mutation frequency, showing the importance of overall
dNTP levels during replication on mutation frequency in vitro. When whole-cell
dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels
underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells. / Graduation date: 2002
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32420 |
| Date | 09 April 2002 |
| Creators | Martomo, Stella A. |
| Contributors | Mathews, Christopher K. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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