Graduate Thesis Or Dissertation
 

The effects of protein associations on pyrimidine deoxyribonucleotide biosynthesis

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  • The faithful replication of DNA depends on the appropriate balance of DNA precursors. From studies conducted in bacteriophage T4, models for deoxyribonucleotide biosynthesis producing pools appropriate for DNA replication have made it possible to understand more complex systems. A portion of that body of evidence supports the concept that deoxyribonucleotide biosynthesis for bacteriophage T4 is carried out by an association of enzymes and other cellular components in a complex called the dNTP synthetase complex. This dissertation explores potential direct protein-protein interactions within this complex for the preparation of pyrimidine deoxyribonucleotides. Direct associations for enzymes involved in pyrimidine deoxyribonucleotide biosynthesis were examined by affinity chromatography. It was determined that there was a significant direct relationship between T4 thymidylate synthase and T4 dCMP deaminase, between T4 dCTPase/dUTPase and T4 dCMP deaminase as well. The interaction between thymidylate synthase and dCMP deaminase was significantly influenced by the presence of dCTP, a positive effector of dCMP deaminase. Furthermore, protein associations changed the kinetic character of pyrimidine deoxyribonucleotide production. T4 dCTPase/dUTPase, a member of the dNTP synthetase complex, significantly alters the kinetic nature of thymidylate synthase by working with thymidylate synthase in a reciprocal relationship. T4 single-stranded DNA binding protein, a member of the replication complex, alters the activity of thymidylate synthase as well. Attempts to isolate a kinetically coupled complex from two or more constituent proteins of the dNTP synthetase complex were frustrated by protein degradation to fragments under 10 kDa in size. Pyrimidine deoxyribonucleotide synthesis is located between the significant energy investment of ribonucleotide reductase and phosphate attachments by kinases to prepare the deoxyribonucleotide molecules for DNA replication. In bacteriophage T4, intermediate reactions are driven by mass action but are modulated by subtleties including direct protein associations and the presence of small molecules that influence enzyme function. Through these and potentially similar controls, pools of deoxyribonucleotides are prepared and delivered in a timely, balanced manner to the DNA replication apparatus.
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