Biochemistry of Deoxyribonucleic Acid-defective Amber Mutants of Bacteriophage T4

Abstract

Abstract Nucleotide pools were analyzed under different conditions of infection by bacteriophage T4, for three reasons: (a) as an approach to identifying the defective functions associated with conditional lethal phage mutants bearing uncharacterized lesions in DNA replication; (b) to gain information about the control of nucleotide synthesis in phage-infected Escherichia coli; and (c) as an aid to interpretation of earlier data (Mathews, C. K. (1968) J. Biol. Chem. 243, 5610) which suggested that the rate of total RNA synthesis decreases when DNA replication is blocked. In infection by wild type T4D there are no significant changes in ribo- or deoxyribonucleoside triphosphate pool sizes, except for replacement of dCTP by 5-hydroxymethyl-dCTP. By contrast, infection by DNA-negative mutants causes up to 30-fold pool expansions of dATP and dTTP, with hydroxymethyl-dCTP accumulating to similar extents. The dGTP pool, however, does not expand significantly, suggesting that the synthesis of this nucleotide is regulated more closely than those of the others. Ribonucleoside triphosphate pools do not expand dramatically in infection by mutants in genes 42, 44, or 62, but up to 5-fold pool expansion is seen in infection by mutants in genes 41 and 45. These studies indicate that the products of genes 41, 44, 45, and 62 do not directly affect the synthesis of DNA precursors. Moreover, comparative studies on the rates of labeling of RNA precursors late in infection support the earlier reported conclusion that the rate of RNA synthesis decreases considerably when DNA synthesis is blocked. Finally, the data allow calculation of the adenylate energy charge and of approximate concentrations of nucleotides in exponentially growing E. coli. The energy charge is about 0.9, in agreement with published work, and this value does not change significantly under the conditions of infection examined. The intracellular concentrations of nucleoside triphosphates range from 0.1 mm for dGTP to 2.7 mm for ATP. These values allow one to ask which of various enzyme feedback effects observed in vitro might reasonably be assumed to be operating in vivo.