Vaccinia virus ribonucleotide reductase : regulation of the gene products and characterization of the recombinant small subunit protein Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/fn1073362

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  • 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.
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  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2013-02-13T21:34:37Z (GMT) No. of bitstreams: 1HowellMeredithL1993.pdf: 37714733 bytes, checksum: 136fd0f3230d8f315448ffa55fa9f5c8 (MD5)
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