Protein-Protein Interactions in DNA Precursor Biosynthesis

Oregon State University, Corvallis OR

Investigators

Abstract

Enzymes catalyzing sequential reactions are increasingly found to be associated within cells. Sometimes the association involves multifunctional proteins, such as those in eukaryotic fatty acid synthetase complexes, sometimes tightly linked enzyme complexes, such as the glycine cleavage system, and sometimes membrane-associated complexes, such as mitochondrial respiratory complexes. Most enzymes, however, are soluble proteins, readily isolated free of other proteins. At the high protein concentrations found within cells, such enzymes can associate into loosely bound complexes, which the late Paul Srere termed metabolons. A complex that has favorable properties for structural and kinetic analysis of the metabolon concept is the bacteriophage T4 dNTP synthetase complex. Previous research from this laboratory has demonstrated a complex containing at least eight phage-coded proteins and two of host cell origin. Individual activities within the isolated complex are kinetically linked. Indirect evidence suggests that the complex is associated with the DNA replication apparatus and that it plays an important role in delivering deoxyribonucleoside triphosphates (dNTPs) to replisomes fast enough to sustain DNA chain at rates of 500-800 nucleotides per second. All of the enzymes known to be in the complex and all T4 DNA replication proteins are available as purified recombinant proteins, allowing numerous approaches to identifying specific protein-protein interactions and analysis of kinetic coupling through studies on partially reconstituted forms of the complex. This project includes the following specific aims: (1) To identify direct and indirect interactions among proteins in the T4 dNTP synthetase complex and interactions with T4 replication proteins shown to associate with the complex. This involves analysis in an optical biosensor, as well as protein affinity chromatography and nondenaturing gel electrophoresis; (2) To define effects of substrates and allosteric effectors upon protein-protein associations. Evidence suggests that small molecules have dramatic effects upon protein associations in the dNTP synthetase complex; (3) To define effects of protein-protein interactions upon kinetic behavior of individual enzymes and linked multi-step reaction pathways. Kinetic analysis of partially reconstituted complexes explores the nature of kinetic coupling. Steady-state analyses, and, in collaboration with a colleague, pre-steady-state analyses using quench-flow techniques are being used; (4) To ascertain whether the dNTP synthetase complex is associated with the replication machinery in vivo. These experiments involve immunolocalization of E. coli dNTP-synthesizing enzymes, specifically, ribonucleotide reductase and thymidylate synthase, and are also being done in a collaborative effort. Crystal structures of some of the enzymes in the complex have been determined and other structure determinations are under way. As structural information about proteins and docking sites emerges, these data will be merged with the enzymological data from this study, in an attempt to develop a unified picture of this particular metabolon.