Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Simon Neubort, Julius Marmur
1973 Journal of Virology  
Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate
more » ... form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component. Bacteriophage SP-15 is a generalized transducing phage infecting sensitive strains (W23, Marburg, and ATCC 6633) of Bacillus subtilis (16, 26). The DNA of SP-15 possesses a number of highly unusual properties: (i) an unusually low thermal denaturation temperature in 0.195 M Na+ (61.5 C); (ii) sensitivity to alkali, prolonged incubation in 0.3 M KOH, causing a gradual degradation of the molecule to fragments sedimenting at 5.5S in alkaline salt solutions; (iii) alkali-induced release of an unknown phosphorylated carbohydrate from the macromolecule, accompanied by a reduction in the buoyant density; and (iv) the replacement of thymine by the unusual pyrimidine base, 5-(4',5'-dihydroxypentyl) uracil (DHPU) (2, 16 ). Our present model of the structure of the DNA molecule shows that DHPU is contained in both strands of the DNA. There is a sugar in a phosphodiester linkage which is presumably linked to the 4' or 5' hydroxyl group, or both, of the side chain of DHPU, and this sugar-phosphate, which is the component responsible for the high density, is removed by incubation in alkali. The identity of this sugar phosphate component is presently under investigation (C. ' Present address:
doi:10.1128/jvi.12.5.1078-1084.1973 fatcat:mxfkw4ijyjbdtftzemdhr6tspm