Graduate Thesis Or Dissertation
 

Enzyme associations in deoxyribonucleotide biosynthesis : anti-idiotypic antibodies as probes for direct protein-protein interactions

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  • The ability to faithfully replicate DNA is dependent upon the maintenance and regulation of its precursors, the deoxyribonucleoside triphosphates. Enzymes encoded by the bacteriophage T4 have been widely used as models of biochemical processes. A body of evidence supports the concept that the bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are associated as a complex within the infected Escherichia coli. This dissertation describes the continued examination of the protein-protein interactions involved in deoxynucleotide biosynthesis of bacteriophage T4. My studies on the protein-protein interactions involved in deoxyribonucleotide biosynthesis focused on two unique phage proteins, the dCMP hydroxymethylase enzyme and the translational regulator RegA. An initial study was undertaken to determine if the generation of anti-idiotypic antibodies would prove useful in the identification of direct interactions between dCMP hydroxymethylase and other proteins of the deoxyribonucleotide synthetase complex. For the initial generation of anti-idiotypic antibodies, polyclonal rabbit antibodies were generated to affinity purified anti-dCMP hydroxymethylase polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the bacteriophage T4 dTMP synthase. A second method to generate anti-idiotypic antibodies was attempted with the translational regulator RegA. The generation of anti-idiotypic antibodies to the RegA protein involved the purification of anti-RegA rabbit Fab fragments and the generation of anti-anti-RegA polyclonal antibodies within mice. This alternative method was determined to be inferior to the initial method for generating anti-idiotypic antibodies. Additional studies involved the examination of RegA protein-protein interactions using affinity chromatography. A number of bacteriophage T4 early proteins were determined to associate with an immobilized RegA column.
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