Properties of Bacteriophage T4 ribonucleoside diphosphate reductase subunits coded by nrdA and nrdB mutants.
As a part of the study of the bacteriophage T4-induced deoxyribonucleotide synthetase complex, an investigation has been made of the T4 ribonucleoside diphosphate reductases formed by a series of mutants of nrdA and B, the genes coding, respectively, for the alpha 2 and beta 2 subunits of the enzyme. dATP affinity columns were used to isolate the enzyme by a single-step procedure. The molecular weights of the alpha and beta chains have been found to be 84,000 and 43,500, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since alpha 2 beta 2 is bound to dATP affinity columns through allosteric effector sites on alpha 2, it is possible to monitor the binding of beta 2 to alpha 2. dTTP- and ATP-Sepharose columns did not bind T4 alpha 2 beta 2, although the corresponding nucleoside triphosphates are effectors of the enzyme and although the alpha 2 subunit of the host enzyme binds to these columns. Missense mutants of nrdA and B forming alpha 2 and beta 2 subunits that lacked catalytic activity but retained the ability to form the alpha 2 beta 2 complex have been described. The 50,000-dalton fragment formed by an amber mutant of nrdA did not bind to the dATP affinity column, providing evidence that a region of the carboxyl-terminal segment of the alpha chain is required for retention. The beta 2 subunit appears to protect the alpha 2 protein. On infection by nrdB mutants not forming beta 2, the alpha protein chain was cleaved specifically to form 3 protein chains of 61,000, 57,000, and 24,500 daltons, which retain the ability to bind to dATP-Sepharose. Some effects of mutation on the interaction of the alpha and beta chains of the enzyme with the deoxyribonucleotide synthetase complex have been examined.
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005814:Genes, Viral; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; C038110:deoxyribonucleotide synthetase complex; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase