Our aim is to understand the nature of the assembly and regulation of the bacteriophage T4-induced deoxyribonucleotide synthetase complex and its coupling to the DNA replication apparatus. These studies seek to elucidate a possible model mechanism switching on deoxyribonucleotide synthesis, and thus T4 DNA, after infection by bacteriophage T4. Our efforts are focused on T4-coded ribonucleoside diphosphate reductase which is the limiting step in the deoxyribonucleotide synthetase complex. A new T4 mutant, nrdB93, encodes a temperature-sensitive protein chain, Beta2, one of the two subunits of T4 ribonucleoside diphosphate reductase. nrdB93 is also defective in the synthesis of its protein, but it is not known whether this occurs at a transcriptional or translational level. The defect in nrdB93 can be demonstrated in vivo by the rate of deoxyribonucleotide synthesis or in vitro by Sepharose-dATP columns and by two dimensional electrophoresis. The poor synthesis of the Beta93 chain is phenotypically suppressed by mutants of gene 39, coding for one of the subunits of T4 DNA topoisomerase, but not by amber mutants of genes 52 and 60 coding for the other two subunits. However, suppression by gene 39 mutants is prevented by a mutation in the host gyrB gene (DNA gyrase). In order to understand these effects we are cloning the T4 nrd genes in bacterial plasmids, and these genes are being sequenced to characterize the gene structure. Both the wild type genes and nrdB93 will be examined for in vitro transcription and translation in their cloned states, and gyrA and gyrB proteins and gene 39 protein will be tested in this system for activation or antagonism of synthesis. himB(gyrB) and himA genes, required for phage Lambda site-specific recombination and phage Mu growth, may also be part of the activation system, and mutants in gyrB, gyrA and in him genes will be tested for Beta93 synthesis in vivo. We also will continue studies on the deoxyribonucleotide synthetase complex by investigating the functional and physical coupling of this complex to the replication enzymes which are contained in our preparations. Phospholipid synthetic vesicles and DNA will be added to our membrane-free, DNA-free preparations of the complex in reconstituting the system. T4 DNA topoisomerase, found in our preparations in large quantities, also binds tightly to the synthetic membranes. A test of the interaction of ribonucleoside diphosphate reductase with DNA is also planned.
