Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs

Abstract

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.

Polyamines in microorganisms.

“…The modification occurs at the polynucleotide level, with label from omithine appearing in the hypermodified base in vivo; in extracts, labeled putrescine is incorporated into the DNA. Studies with amber mutants indicate that a portion of the a-putrescinylthymine is essential for the production of viable phage, probably because of its effect on DNA packaging in the phage head (96,107,108,118,182).…”

α-Putrescinylthymine and the sensitivity of bacteriophageφW-14 DNA to restriction endonucleases

“…The release of discrete fragments from em42rev4 and amh2 DNAs, in which the putT contents are 6 and 9 moles percent, respectively, shows that the distribution is ordered in these DNAs, too. Thus, the product of the gene defined by aj42 determines, or is intimately involved in, the sequence-specific conversion of hmU to putT at the polynucleotide level (15,37). The mutation in sm42 obviously reduces, but does not altogether eliminate, the sequence specificity of the modification reactions.…”

“…am42 is a mutant of pW-14 that, in the non-permissive host, produces DNA containing 6 moles percent putT rather than the 12 moles percent found in the wild-type (15); this DNA is packaged into particles which are infectious but unable to produce progeny (15). am42rev4 is a revertant of am42 that produces progeny in the non-permissive host; its DNA contains 9 moles per cent putT (15). Phage preparations were purified by differential centrifugation.…”

“…The substituent on jW-14 DNA is smaller than that on SP15 DNA, however, and it is positively rather than neg-atively charged. The positive charges on putT can be removed by acetylation (13), and mutants of jW-14 have been isolated in which the putT content of the DNA is reduced relative to that of wild-type DNA (14,15). These different forms of SW-14 DNA can be used to examine both charge and steric effects of putT on the sensitivity of the DNA to Type II enzymes.…”

DNA synthesis in Pseudomonas acidovorans infected with mutants of bacteriophage phi W-14 defective in the synthesis of alpha-putrescinylthymine

“…am42 produces particles in strain 29 which are infectious but incapable of producing further progeny because the DNA molecules they contain are about 11% shorter than those in wild-type particles, a consequence of their lower content of putThy (10). Such particles directed the synthesis of DNA in strain 3L.…”