Graduate Thesis Or Dissertation
 

Host kinases involved in DNA precursor biosynthesis during bacteriophage T4 infection

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  • Although the Escherichia coli host has almost all of the enzymes necessary to synthesize nucleotides needed for bacteriophage T4 DNA replication, phage genes expressed early in infection encode enzymes for de novo DNA precursor biosynthesis and salvage from degraded host DNA. Eight early enzymes and two host enzymes comprise the multienzyme dNTP synthetase complex. The complex utilizes two host kinases for phage replication: nucleoside diphosphate kinase (Ndk) and adenylate kinase (Adk). The dNTP synthetase complex and the replication apparatus interact in vivo. dNTP synthesis is kinetically coupled to T4 DNA synthesis in a wild-type host but not in an ndk host. Moreover, one indirect and four direct experimental approaches demonstrate partial reconstitution of interactions between the two complexes in vitro. These interactions include Ndk and T4 DNA polymerase, which catalyze consecutive metabolic steps (dNTP synthesis and replication). The effect of the ndk mutation on the E. coli host was also studied. Disruption of the host ndk gene has been reported to cause a mutator phenotype due to nucleotide pool imbalances from a hugely increased dCTP pool. The pool imbalance has little effect on growth rate, since the ndk strain growth rate is 5.8 min. (15%) slower. However, the rate of phage DNA synthesis is reduced by 83.7% in the ndk strain relative to its parent. Although Adk complements ndk disruption, Adk's NDP kinase activity has high KM values for dNDP substrates. Adk has a novel (dNTP:AMP) phosphotransferase activity which synthesizes the dNTP's for replication. As measured by selectivity (kcat/KM), Adk's (dNTP:AMP) phosphotransferase activity--not its NDP kinase activity--is the physiologically relevant mechanism for dNTP synthesis in the ndk mutant. Therefore, ADP is the physiological phosphate donor instead of ATP. The ndk strain was found to have abnormally elevated nucleotide pools of UTP, CTP, dCDP and dTTP. In the ndk mutant, a high activity of Adk upon UDP leads to excess UTP production. Accompanied by a 3.2-fold increase in CTP synthetase activity, subsequent nucleotide pools are also increased, culminating in 18-fold dCTP and 2.4-fold dTTP pool accumulations, and hence, the mutator phenotype.
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