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A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6-fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper. [ABSTRACT FROM AUTHOR]

The effects of nucleotide analogues on DNA synthesis ere studied in nucleotide permeable Escherichia coli cells. 2′,3′,Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In ϕX174-infected cells this analogue caused the formation of short pieces (7 S) of ϕX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of ϕX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coil DNA pieces. Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of ϕX replicative form replication, nor was evidence obtained that nuoleoside monophosphates are immediate precursors of DNA. [ABSTRACT FROM AUTHOR]