| Summary: | Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting
step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project
was to characterize the ribonucleotide reductase encoded by the orthopoxvirus,
vaccinia. The first objective was to study the structural and functional features of the
viral small subunit protein of ribonucleotide reductase. The viral reductase gene was
engineered into an expression vector and expressed in Escherichia coli. The purified
recombinant protein was then characterized and compared with other ribonucleotide
reductase small subunits from different organisms. The physical characteristics of the
vaccinia virus enzyme showed a strong similarity to the features of the mammalian
counterpart.
A second aim of this project was to establish the transcriptional and translational
kinetics of ribonucleotide reductase gene expression during the time course of viral
infection in cultured mammalian cells. In addition, the activity and stability of the
enzyme in the viral system was measured and the accumulation of ribonucleotide
reductase protein was quantitated. By also quantitating the accumulation of viral DNA
synthesis, a direct comparison can be made between the the synthesis and utilization of
deoxynucleotide precursors.
A third objective of this work was to detail the mechanism by which
hydroxyurea inactivates the vaccinia virus ribonucleotide reductase. Visible
spectroscopy and electron paramagnetic resonance spectroscopy clearly demonstrated
that the inhibitor destroys the free radical moiety in the viral small subunit protein. In
addition, in vivo studies revealed that inhibition by hydroxyurea can be circumvented
during viral infection. The exogenous addition of deoxyadenosine reversed the block
to viral growth that was imposed by hydroxyurea, and stabilized hydroxyurea induced
deoxynucleotide pool imbalances. These inhibition studies suggest that there may be a
differential sensitivity of the enzyme towards hydroxyurea in the presence of various
substrates. === Graduation date: 1993
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