Abstract:
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.
