In order to maintain genetic stability in eukaryotes, tight regulation of the
relative sizes of deoxyribonucleoside triphosphate (dNTP) levels inside the cell is
essential for optimal fidelity of DNA replication. Ribonucleotide reductase (RNR)
is the enzyme responsible for proportional production of DNA precursors. Studies
on regulation of this enzyme, the focus of this thesis, are important because
mutations affecting RNR control mechanisms result in dNTP pool imbalance, thus
promoting mutagenesis.
By using mouse RNR as a model for mammalian forms of the enzyme,
three major factors--allosteric effectors, rNDP substrate concentrations, and
hypoxic conditions--that influence the substrate specificity of RNR have been
investigated. Allosteric regulation has been studied by the four-substrate assay,
which permits simultaneous monitoring of the four reactions catalyzed by this
enzyme in one reaction mixture. Individual dNTPs affect the four activities
differentially in a concentration-dependent manner with discrete effects of dTTP
and dGTP on reduction of ADP and GDP, respectively. Ribonucleoside
diphosphate (rNDP) substrate concentrations are equally important, as their
variations lead to different product ratios. Results from nucleotide binding assays
indicate that rNDPs directly influence binding of dNTP effectors at the specificity
site, one of the two classes of allosteric sites, whereas ADP has an indirect effect,
displacing other substrates at the catalytic site and consequently removing effects
of those substrates upon dNTP binding. Hence, this is the first evidence of a two-way
communication between the catalytic site and the specificity site. Oxygen
limitation also plays an important role in controlling the enzyme specificity.
Reactivation of the enzyme at different oxygen tensions, after treatment of the
enzyme with hydroxyurea (HU) followed by removal of HU, reveals a distinct
sensitivity of GDP reductase to low 0��� levels. Although the basis for specific
inhibition of GDP reduction remains to be determined, some possibilities have been
ruled out.
This research proves that in addition to allosteric regulation by nucleoside
triphosphates, mouse RNR is also controlled by other factors. Since these
components can simultaneously exert their effects upon enzyme specificity,
complex regulatory patterns of RNR to provide a proportional supply of the DNA
building blocks in vivo are suggested. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31161 |
| Date | 12 June 2002 |
| Creators | Chimploy, Korakod |
| Contributors | Mathews, Christopher K. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0022 seconds