A crucial factor in determining the accuracy of DNA replication is
maintenance of a balanced supply of deoxyribonucleoside triphosphates
(dNTPs) at replication forks. Perturbation of dNTP biosynthesis can
induce dNTP pool imbalance with deleterious genetic consequences,
including increased mutagenesis, recombination, chromosomal
abnormalities and cell death. Using the T4 bacteriophage system, I
investigated the molecular basis of mutations induced by imbalanced
dNTP pools in vivo. Two approaches were adopted to disturb dNTP
biosynthesis: 1) using mutations which affect the deoxyribonucleotide
biosynthesis pathway; 2) exogenously supplying mutagenic
deoxyribonucleoside analogs which are then taken up by cells and are
metabolized to dNTPs. The levels of dNTPs under different conditions
were measured in crude extracts of phage-infected cells, while
mutagenic effects were quantitated by analysis of certain rII mutations,
thought to revert to wild type along either GC-to-AT or AT-to-GC
transition pathways. The mutation pathways stimulated by dNTP pool
perturbations were confirmed by direct DNA sequencing after
amplification of template by the polymerase chain reaction (PCR).
By replacing phage ribonucleotide (rNDP) reductase with the host,
Escherichia coli, rNDP reductase, in phage-infected cells, I examined
the mechanism of mutation induced by the thymidine analog 5-
bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to-
AT transition mutations were stimulated many hundred-fold when cells
were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions
were stimulated predominantly when T4 reductase was active, while ATto-
GC transitions were stimulated more when E. coli reductase was
active. By examining the control by dNTPs on CDP reduction, I found that
the T4 rNDP reductase is substantially inhibited by either BrdUTP or
dTTP in crude enzyme extracts. These experimental results are
consistent with the hypothesis that mutagenic effects of BrdUrd are
based on dNTP perturbations, supporting the model that rNDP reductase
is a major determinant of BrdUrd mutagenesis.
I also studied the mutator phenotype of one temperature-sensitive
conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4
deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII
mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions
induced by ts LB3 at a semipermissive temperature (34° C). Sequence
analysis of revertants from the most sensitive gene marker, rII SN103,
showed that either cytosine within the mutated triplet can undergo
change to either thymidine or adenine, supporting a model in which
mutagenesis induced by ts LB3 at a semipermissive temperature is based
on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine
triphosphate (hm-dCTP) caused by the temperature-labile
dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP
and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T
transition and C-to-A transversion mutations. However, no significant
dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected
cells even when cells were grown at the semi-permissive
temperature, suggesting that imbalanced dNTP pools occurred only
locally, close to replication forks. These results support a model of dNTP
"functional compartmentation", in which DNA replication is fed by a small
and rapidly depleted pool, with the bulk of measurable dNTP in a cell
representing a replication-inactive pool.
To further characterize the mutagenic specificity and DNA site
specificity induced by T4 ts LB3, I developed a fast forward mutation
approach using thymidine kinase as a marker gene. The studies
confirmed that the principal mutagenic effect induced by ts LB3 is C-to-
T transition, while C-to-A transversion mutagenesis also occurs. Analysis
of DNA sequences around each mutation also suggests that local DNA
context influences mutation frequency. / Graduation date: 1991
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36993 |
| Date | 02 November 1990 |
| Creators | Ji, Jiuping |
| Contributors | Mathews, Christopher K. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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