T4 Phage deoxyribonucleoside triphosphate synthetase: Purification of an enzyme complex and identification of gene products required for integrity

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Abstract
We have isolated a highly enriched preparation of the multienzyme complex which synthesize Deocyribonuleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparation. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exits in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene product encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.