Single-Strand DNA-Binding Proteins and Deoxyribonucleotide Enzyme Organization

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9603684 Mathews This project tests a model of DNA replication in which the deoxyribonucleoside triphosphate precursors to DNA are synthesized by enzyme complexes linked to the replication machinery, allowing thereby the efficient delivery of building materials to DNA replication sites. In T4 bacteriophage-infected Escherichia coli, the enzymes of dNTP biosynthesis form a complex, called dNTP synthetase, which contains at least eight virus-coded enzymes and two enzymes of host cell origin. In vitro, the complex displays kinetic coupling, i.e., the ability to catalyze multi-step reaction pathways efficiently and with little accumulation of intermediates. Thus, the complex behaves in vitro as it must behave in vivo if it is to serve as a DNA substrate shuttle, synthesizing dNTPs in the immediate vicinity of replication sites. Purified recombinant enzymes from the T4 dNTP synthetase complex have been shown to associate, directly or indirectly, with the T4 single-strand DNA-binding protein(SSB) encoded by gene 32. This suggests a model for DNA replication in which multiple dNTP synthetase complexes are drawn to replication forks by their attraction for T4 SSB. This project will test that model by physical and enzyme kinetic analysis of association among proteins in the complex and the T4 gene 32 protein; investigations of the role of gene 32 protein in drawing dNTP synthesizing enzymes to replication sites; investigation of the role the acidic C-terminus of gene 32 protein upon interactions with dNTP-synthesizing enzymes; and attempted reconstitution of parts of the dNTP synthetase complex. If resources permit, this investigation will be extended to vaccinia virus, which also encodes an SSB that interacts with enzymes of DNA precursor synthesis. Many cellular metabolic processes are being found to involve organized protein assemblies, which increase the efficiency of cellular function by preventing diffusion of intermediates away from sites of utilization. Because DNA replication occurs at a limited number of sites (as few as two replication forks per chromosome in bacteria), there is a special need for organization of enzymes of dNTP synthesis, to deliver precursors to those few sites rapidly enough to support observed high rates of DNA replication and to maintain high accuracy in the transfer of genetic information. Moreover, because dNTPs have no known metabolic roles other than their use as DNA precursors, the synthesis of those precursors must be closely coordinated with DNA replication. Understanding that coordination may lead to a greater understanding of the intracellular organization of a fundamental biological process.