This proposal will study the coordination between DNA replication and the synthesis of its precursors, the deoxyribonucleoside triphosphates (dNTPs). In T4 phage-infected E. coli, the enzymes of dNTP biosynthesis form a complex, called dNTP synthetase, which contains at least eight viral-encoded and two host-cell enzymes and which shows kinetic coupling in vitro. The physical relationship between the dNTP synthetase complex and the multi-protein DNA replication complex has not been defined, and that relationship represents a main thrust of this proposal. Eight of the enzymes in the complex have been purified as recombinant proteins and immobilized on chromatographic column. Analysis of T4 proteins bound shows that each column retains gp32, the single-stranded DNA-binding protein (SSB) encoded by T4 gene 32. A simplifying idea is that gp32 acts as a connector, drawing the dNTP synthetase complex to the replication fork, possibly in multiple copies of dNTP synthetase per fork. Since gp32 binds single-stranded template DNA strands just ahead of the daughter DNA 3'-hydroxyl terminus, this association would place dNTP synthetase precisely where it is needed to saturate the space where DNA replication is occurring. A similar situation may exist in cells infected with vaccinia virus, where a single-strand-specific DNA-binding protein that interacts with viral-encoded ribonucleotide reductase has been described. Affinity chromatography with immobilized proteins and DNA-cellulose, non-denaturing gel electrophoresis, and immunoprecipitation will be used to distinguish those proteins in the dNTP synthetase complex that interact directly with gp32 from those that interact indirectly. The effects of purified gp32 upon physical and kinetic association among dNTP synthetase enzymes will be probed. Surface plasma resonance will be analyzed to quantitate binding affinities, stoichiometry, and cooperativity. Perturbing gene 32 function in vivo will be tested for its effects on the association of dNTP synthetic enzymes with DNA-protein complexes isolated from infected cells. To test the general applicability of the model, the vaccinia virus SSB will be further analyzed. Development of a conditional expression system will permit experiments to define the metabolic roles of the protein, in supporting DNA replication and possibly in drawing viral-encoded dNTP synthetic enzymes to replication sites. Affinity chromatography, of immobilized p34 and its C-terminal truncated derivatives, will permit identification of its protein associations. Characterization of the phosphorylation of SSB will explore the role of this modification on protein-protein and protein-DNA associations.
