Synthesis of 5-hydroxymethyldeoxyuridine triphosphate in extracts of SP10c phage-infectedBacillus subtilis W23

Mature DNA of Bacillus subtilis W23 phage SP10 contains a hypermodified nucleotide (YdTMP) that replaces ca. 20% of the dTMP. SP10 DNA was pulselabeled for 1 min at 200C with 32Pi. Among the oxopyrimidine nucleotides, virtually all of the radioactivity was recovered as 5-hydroxymethyldeoxyuridylate (HMdUMP). During a subsequent chase, radioactivity was lost from HMdUMP and recovered as YdTMP and dTMP. At 370C, exogenous [6-3H]5-hydroxymethyldeoxyuridine (HMdUrd) was incorporated into SPIO DNA. Label administered as HMdUrd was phosphorylated to HMdUTP in the infected cells, but all radioactivity was recovered from SP10 DNA as YdTMP and dTMP. Two heatsensitive mutants defective in hypermodification of SP10 DNA are described. In one mutant, HMdUMP replaces YdTMP in DNA. The other mutant generates a DNA containing a novel deoxynucleotide in place of YdTMP. The novel deoxynucleotide seems to consist of PPi esterified to the 5-hydroxymethyl function of HMdUMP (PP-HMdUMP). Both mutants make normal amounts of dTMP. The data are discussed in terms of the following conclusions. (i) Both oxopyrimidine nucleotides in mature SP10 DNA are derived by postreplicative modification of HMdUMP in nascent DNA. (ii) PP-HMdUMP is an intermediate that facilitates formation of a putative exocyclic methylene intermediate which receives the hypermodification. It is also argued that PP-HMdUMP and the same exocyclic methylene intermediate could serve as intermediates in reductive modification to dTMP. (iii) YdTMP is not an intermediate in the formation of dTMP, and reductive modification proceeds independently of hypermodification.

mentioning

confidence: 99%

mentioning

confidence: 99%

Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate

Witmer¹

1981

“…At temperatures of < 30°C, mutant (hmd) phage gave bursts that were within 60% of normal, but at higher temperatures the burst size declined sharply (Table 1). For this paper, hmd+ and hmd phage were compared at 37°C since this is the temperature at which most biochemical studies with SP10 were performed (10,29,30,39,40). SPno hmdl 20 54 12 sl x 10-4 sl x 10-4 SP10 hmd2 21 58 12 sl x i-0 sl x i0-4…”

Section: Resultsmentioning

confidence: 99%

DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10

Witmer

Franks

1982

Self Cite

A hypermodified base (Y-Thy) replaces 20%o of the thymine (Thy) in mature DNA of Bacillus subtilis phage SP10. Two noncomplementing hypermodificationdefective (hmd) mutants are described. At 30°C, hmd phage carried out a normal program, but at temperatures of .37TC, the infection process was nonproductive. When cells were infected at 37°C with hmd phage, DNA synthesis started at its usual time (12 mmin), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under hmd conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodifled nascent strands. The reduced levels of DNA synthesis on September 25, 2020 by guest http://jvi.asm.org/ Downloaded from ROLE OF Y-Thy IN SP10 DEVELOPMENT 637 seems to be required for proper replication of the phage genome as well as cleavage of concatenates to unit-size DNA. MATERIALS AND METHODS Phage and bacteria. SP10 hmdl and SP10 hmd2 are heat-sensitive mutants defective in hypermodification (see below and reference 39). SP10 hmd+ is the same clear-plaque variant used in all previous studies (29, 30). B. subtilis W23 was the host in all experiments. Mutagenesis. Cells infected with SP10 hmd+ at 30°C were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine as described by Kahan (20). Screening for hmd mutants. Survivors of mutagenesis were plated at 30°C. Plaques were picked with sterile toothpicks and scored for their ability to plate at 42°C. Lysates of heat-sensitive mutants were prepared at 30°C and used to infect cells at 42°C in the presence of 6-(p-hydroxyphenylazo)uracil, an inhibitor of bacterial DNA polymerase III (5, 32) that has no remarkable effect on SP10 development (29, 30). The cells were labeled with [6-3H]uracil (10 uCi/ml) for 15 min. Cellular DNA was isolated (6) and acid hydrolyzed to a mixture of purine bases and pyrimidine deoxynucleosides (36). The digests were chromatographed in one direction on unmodified cellulose thin layers (10), using deoxythymidine (dThd) and Y-dThd as optical markers. Spots corresponding to the optical markers were eluted (10, 29), and the amount of label present was determined. In those cases where no label was recovered in the Y-dThd spot, a sample of [3H]DNA was enzymatically degraded to mononucleotides (36, 39) that were fractionated by two-dimensional chromatography on unmodified cellulose thin layers (7). Of the 184 single-plaque isolates tested to date, only 2 generated DNA at 42°C which was totally devoid of Y-dThd. The bases replacing Y-Thy in SP10 hmdl and SPO hmnd2 DNAs, under nonpermissive conditions, are 5-(hydroxymethyl-O-pyrophosphoryl)uracil and

“…In SP10 DNA, 15 to 20% of the thymine is replaced with a base carrying a secondary amine function at C-5 of the uracil ring (M. Mandel, personal communication). The thymine is formed at the polynucleotide level, probably from hmUra (3,18). Infection with SP10 also leads to the inhibition of host thymidylate synthase (18) and to the appearance of dTTPase (12) and dUMP hydroxymethylase (18) activities; hmdUTP is also found in the infected cells (3).…”

Section: Discussionmentioning

confidence: 99%

“…The thymine is formed at the polynucleotide level, probably from hmUra (3,18). Infection with SP10 also leads to the inhibition of host thymidylate synthase (18) and to the appearance of dTTPase (12) and dUMP hydroxymethylase (18) activities; hmdUTP is also found in the infected cells (3). It is of considerable interest that the two phages, one attacking a gram-negative host and the other attacking a gram-positive host, should be so similar in their biochemistry.…”

Section: Discussionmentioning

confidence: 99%

Bacteriophage phi W-14-infected Pseudomonas acidovorans synthesizes hydroxymethyldeoxyuridine triphosphate

Neuhard¹,

Maltman²,

Warren³

1980

The infection of Pseudomonas acidovorans with bacteriophage phi W-14 leads to the gradual disappearance of dTTP from the cells and to the appearance of hydroxymethy dUTP (hmdUTP). Infected-cell contain dUMP hydroxymethylase and activities converting hmdUMP to humdUDP and hmdUTP. Hydroxymethylase appears immediately after infection, reaching a maximum 20 min later. Thymidylate synthase activity decreases to less than 10% of the preinfection level during the initial 40 min after infection. Newly replicated DNA contains 2 to 3% hydroxymethyluracil. Although uracil is released from newly replicated DNA by acid hydrolysis, uracil is not incorporated as such into phi W-14 DNA, and dUTP is not present in the acid-soluble pool of infected cells. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level and that an intermediate in one or both of these conversions is degraded to uracil by acid hydrolysis. The modification of hydroxymethyluracil is coupled tightly to replication.