Biosynthesis of 5-(4'5'-dihydroxypentyl) uracil as a nucleoside triphosphate in bacteriophage SP15-infected Bacillus subtilis

Abstract

The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU. (Bausch & Lomb Spectronic 20). Materials. Bases, nucleosides, and nucleotides were obtained from Calbiochem. Si nuclease was purchased from Miles Laboratories, Inc., and snake venom phosphodiesterase came from Worthington Biochemicals Corp. DNase I and Ti RNase were from Calbiochem, and pancreatic RNase A was from Sigma Chemical Co. 6-(p-Hydroxyphenylazo)uracil was a gift from B. W. Langley of Imperial Chemical Industries, Ltd. CsCl was obtained from Metallgesellschaft A.G., Frankfurt, West Germany. [6-3H]uracil, 32Pi, [5,6-3H]uridine, and [methyl-3H]thymidine were supplied by Schwarz/Mann.

Production and expression of dTMP-enriched DNA of bacteriophage SP15

“…Glucose-i-phosphate confers an unusually high buoyant density on SP15 DNA (22). In CsCl, SP15 DNA has a buoyant density of 1.762 g/ml (22,24,36), whereas poly(dGMP-dCMP) has a density of 1.760 g/ml (18). When SP15 DNA is incubated in 0.3 N KOH, glucose-l-phosphate is removed and the buoyant density declines to 1.703 g/ml (22).…”

“…Originally, it was thought that DPHdUMP arose by post-replicational modification of dUMP in nascent DNA (24). Recently, however, DHPdUTP has been identified in formic acid extracts of SP15 phage-infected cells, implying that the hypermodified nucleotide is, in fact, synthesized de novo (36).…”

Incorporation of label from ribose into 5-(4',5'-dihydroxypentyl) uracil of bacteriophage SP15 DNA

“…The base is further modified with three glucoses, one of which is phosphodiester linked (C. Brandon, Ph.D. thesis, Yeshiva University, New York, N.Y., 1974). The detection of DHPU as a nucleoside triphosphate in extracts of phage-infected cells has led to the suggestion that DHPU is synthesized before its incorporation into bacteriophage DNA (6). However, the biosynthetic pathway is unknown.…”

“…8ubtlis W23 was inoculated into NLM medium containing 0.5% D-ribose and no glucose, and when the culture had reached an optical density of 0.6 at 550 nm, it was infected with SP15 at a multiplicity of 5. Forty minutes after infection, the cells were collected by centrifugation and suspended in NLM medium containing 0.1% D-glucose and no ribose. Shaking was resumed, and 10 min later D-[1-14C]ribose (60.2 mCi/mmol; Amersham/ Searle) and [5,6-3H]uracil (9.2 Ci/mmol; ICN Pharmaceuticals, Inc.), each at 2.5 Ci/ml, were added. Thirty minutes after addition of the ra-dioactivity, the cells were harvested, and the DNA was isolated, digested with Ti and pancreatic RNases, reisolated, and enzymatically digested to its deoxynucleoside monophosphates as described elsewhere (6).…”

“…Shaking was resumed, and 10 min later D-[1-14C]ribose (60.2 mCi/mmol; Amersham/ Searle) and [5,6-3H]uracil (9.2 Ci/mmol; ICN Pharmaceuticals, Inc.), each at 2.5 Ci/ml, were added. Thirty minutes after addition of the ra-dioactivity, the cells were harvested, and the DNA was isolated, digested with Ti and pancreatic RNases, reisolated, and enzymatically digested to its deoxynucleoside monophosphates as described elsewhere (6). The desalted digest was chromatographed by high-prmeure liquid chromatography on Partisil SAX (6); the results are shown in Fig.…”