Production and expression of dTMP-enriched DNA of bacteriophage SP15

Abstract

Normal DNA of Bacillus subtilis phage SP15 contains approximately equimolar quantities of dTMP and a hypermodified nucleotide, 5-dihydroxypentyl-dUMP (DHPdUMP). Deoxythymidine (dThd) rescue of phage DNA synthesis in 5-fluorodeoxyuridine (FUdR)-inhibited cultures resulted in the synthesis of SP15 DNA containing enhanced levels of dTMP and correspondingly reduced levels of DHPdUMP. This rescued system was used to probe possible roles of DHPdUMP in phage development. The results suggested that normal levels of DHPdUMP were not required for proper transcription of phage DNA, but normal amounts of DHPdUMP were indispensable for phage assembly and/or DNA maturation. The amount of exogenous dThd required to rescue phage DNA synthesis in FUdR-inhibited cultures was 20-fold higher than the concentration required to rescue cellular replication, whereas the same low concentrations of dThd sufficed to rescue viral and bacterial DNA syntheses in aminopterin-inhibited cultures. Normal SP15 DNA was made in rescued, aminopterin-inhibited cultures. We suggest that FUdR (but not aminopterin) partially suppresses biosynthesis of the hypermodified nucleotide and that there is a barrier to replacement of DHPdUMP by dTMP; therefore, exceptionally large amounts of dThd must be salvaged in FUdR-inhibited cultures to force replacement of the unusual nucleotide by dTMP.