Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4

Abstract

Abstract An in vivo assay of enzyme activity has been employed to investigate the mechanism of control of enzymes synthesizing deoxyribonucleotides after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates into water. At least two phage-induced early enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase, which are synthesized within a few minutes after infection as measured in extracts, do not function in vivo immediately after their formation. Instead, in vivo these enzyme activities initiate about 5 min after infection at 30°. The activities initially increase exponentially, and then become linear. This exponential activation process does not require concomitant protein synthesis. A number of experiments rule against substrate limitation, feedback, or isotope dilution as an explanation of these kinetic data. Thus, the same kinetic behavior in vivo is observed on infection by mutants which are unable to synthesize T4 DNA (DO) and accumulate deoxyribonucleotides. However, amber mutants of gene 43, the structural gene for T4-induced DNA polymerase, are exceptions. Infection by these mutants clearly showed a reduced initial rate of 3H release. Concurrent measurement of DNA synthesis and of in vivo enzyme activity indicates that these two processes follow the same kinetics and coincide in time. These results suggest that the limiting factor in DNA replication is the rate of formation of the deoxyribonucleotide substrates. From these and previous studies we have postulated that dCMP hydroxymethylase and thymidylate synthetase and apparently other enzymes forming deoxyribonucleotides must, to be active in vivo, become part of a complex. We propose that the exponential activation process represents the formation of this complex from its components.