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.

In a nonpermissive host, an amber mutant, am 37, of bacteriophage phi W-14 synthesizes deoxyribonucleic acid (DNA) of considerably greater buoyant density than the DNA synthesized by wild-type phage. The am 37 DNA lacks the hypermodified pyrimidine, alpha-putrescinylthymine (putThy). Instead, it contains a new modified base, 5-[(hydroxymethyl)-O-pyrophosphoryl]uracil (hmPPUra). Extracts of cells infected with wild-type phi W-14 convert the hmPPUra in am 37 DNA to putThy when incubated with putrescine.

Host DNA synthesis stopped about 10 min after the infection of Pseudomonas acidovorans with bacteriophage 4W-14, but host DNA was not degraded to acidsoluble fragments. The synthesis of host but not of phage DNA was inhibited by 5-fluorodeoxyuridine. The nucleotide pools of infected cells did not contain dTTP, and infection resulted in the appearance of dTTPase activity. Although ornithine labeled the a-putrescinylthymine residues of 4W-14 DNA, ornithine-labeled nucleotides were not detected in infected cells. A new deoxynucleoside triphosphate did appear in infected cells, but it was not labeled by ornithine. It is concluded that the thymine and a-putrescinylthymine in OW-14 DNA are synthesized at the polynucleotide level.

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.

Of 42 amber mutants of bacteriophage 4W-14, 6 were defective in DNA synthesis. Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA. The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less a-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no a-putrescinylthymine, and hydroxymethyluracil. The properties of these mutants suggested that the presence of the normal amount of a-putrescinylthymine in 4W-14 DNA was essential for the production of viable progeny. Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host. These mutants were analogous to the DNA off mutants of T4. Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or a-putrescinyldeoxythymidine triphosphate, confirming that both thymine and a-putrescinylthymidine in 4W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level. The synthesis of XW-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming a-putrescinylthymine is essential, and (iii) thymine and a-putrescinylthymine must be made in the correct proportions. Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification. Bacteriophage 4W-14 (7) is unusual because