Organization of T4 bacteriophage genes and gene products involved in DNA precursor biosynthesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/qj72p986q

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  • Bacteriophage T4 gene 42 encodes dCMP hydroxymethylase, an enzyme unique to the deoxyribonucleotide metabolism of T-even bacteriophages. To study biochemical and biophysical properties of the enzyme, as well as the interaction of dCMP hydroxymethylase with other DNA precursor biosynthetic enzymes in vitro, availability of large amounts of the enzyme is necessary. Therefore, cloning and overexpression of T4 gene 42 are needed for these studies. An 1.8-kb Eco RI restriction fragment of a T4 multiple mutant BK536, containing an amber mutation in gene 42, has been cloned into pUC19 plasmid vector in Escherichia. coil. Genetic and biochemical examination of the cloned T4 DNA fragment has revealed that it contains the entire gene 42 coding sequence. The cloned amber mutant gene was converted to a wild type gene by site-directed mutagenesis, and the wild type gene 42 was overexpressed with the pT7 expression vector system. dCMP hydroxymethylase expressed from the cloned gene 42 was purified to homogeneity. The specific activity and N-terminal amino acid sequence of the purified enzyme were determined. The specific activity of the purified cloned gene product closely agreed with the value of the enzyme purified- from phage-infected E. colt cells. Nterminal amino acid determination has confirmed the open reading frame of gene 42, deduced from nucleotide sequence of gene 42. Purified dCMP hydroxymethylase has also been used to study its crystal structure and catalytic mechanism by collaboration with other research groups. In addition, purified dCMP hydroxymethylase was immobilized on Affi-Gel to prepare an affinity column, which was used to study protein-protein interaction among dNTP biosynthetic enzymes and with other DNA metabolic proteins. The proteins which specifically interact with dCMP hydroxymethylase were identified by SDS polyacrylamide gel electrophoresis followed by fluorography, Western blotting, and two dimensional gel electrophoresis. It was earlier found that T4-encoded thymidylate synthase and dihydrofolate reductase function not only in deoxyribonucleotide biosynthesis but are also structural components of the phage baseplate. Two deletion mutants containing deletion in the td gene, encoding thymidylate synthase, and the frd gene, encoding dihydrofolate reductase, were carefully characterized, and used to reevaluate structural role of these two enzymes.
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