This laboratory has suggested that in phage T4 deoxyribonucleotide synthetase, occurs in a complex of enzymes, T4 deoxyribonucleotide synthetase, and that T4 DNA plays a functional role in the complex. The proposed study will investigate the relationships of phage T4 DNA topoisomerase and of DNA to deoxyribonucleotide synthesis. On mutation of the structural genes coding for topoisomerase deoxyribonucleotide synthesis is greatly diminished. We now have found that the activity of the phage-coded ribonucleoside diphosphate reductase in extracts of cells infected by such a mutant is markedly lower than in wild-type infection. By using dATP-Sepharose affinity columns the defect was shown to be in the binding of the alpha2 to the beta2 subunits to form the active enzyme alpha2beta2. Since this association is spontaneous in WT extracts at the concentrations employed and since the nrd genes coding for this enzyme in the mutant are normal, we propose that one of the chains is defective, either in folding or a modification of the primary structure. At the same time we will be studying the interactions of DNA topoisomerase and DNA with the defective subunit to develop a system to convert it to an active subunit. We have isolated an enzyme complex containing T4 ribonucleotide reductase and all of the other components necessary to convert the 4 rNDP species to their dNTP derivatives. In the absence of the beta2 subunit of reductase, or on infection with a mutant defective in DNA topoisomerase, the alpha2 dimer does not enter the complex. Instead, a new complex of alpha subunits with 3 specific phage-coded proteins is formed in the absence of the beta subunit. We propose to study the role of the assembly of ribonucleoside diphosphate reductase in the activation of the deoxyribonucleotide synthetase complex.
Our aim i s to understand the structure and function of the deoxyribonucleotide synthetase and the role played by DNA and the topoisomerase in its assembly and activity. Ultimately we want to couple this complex with the DNA-replication complex, since our in vivo experiments indicate that the two complexes are intimately associated.
