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.

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.     

Synthesis of thymine and alpha-putrescinylthymine in bacteriophage phi W-14-infected Pseudomonas acidovorans.

Host DNA synthesis stopped about 10 min after the infection of Pseudomonas acidovorans with bacteriophage phi W-14, but host DNA was not degraded to acid-soluble 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 alpha-putrescinylthymine residues of phi W-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 alpha-putrescinylthymine in phi W-14 DNA are synthesized at the polynucleotide level.     

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

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.     

Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA.

The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973). Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B. subtilis. B. subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake. Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake. Phi W-14 DNA did not alter the kinetics of transfection. The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA. If failed to inhibit when added 30 min after SPP1 DNA. It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA. The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA. Free putrescine did not affect transfection by SPP1 DNA. It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.     

Alkali lability of bacteriophage phi W-14 DNA.

The molecular weight of bacteriophage phi W-14 DNA, determined by velocity sedimentation in neutral sucrose gradients, was 92 +/- 6 X 10(6). The DNA showed marked fragmentation in alkaline sucrose gradients. This fragmentation was not a consequence of preexisting single-strand interruptions in the DNA, since thermal denaturation of DNA yielded intact single strands. The alpha-putrescinylthymine groups in phi W-14 DNA appeared to be labile; some, or parts of some, of these groups were cleaved from the DNA in alkali.     

Biological Activity of 5-Hydroxymethyluracil and Its Deoxynucleoside in Noninfected and Phageinfected Bacillus subtilis

(14)C-hydroxymethyldeoxyuridine (dHMU) is specifically incorporated into the deoxyribonucleic acid (DNA) of bacteriophage SP8. Incorporation experiments demonstrate that the initiation of phage SP8 DNA synthesis occurs between 12.5 to 15 min after infection. Incorporation into host DNA does not occur. (14)C-dHMU can be used as an analytical tool for screening conditionally lethal phage mutants containing hydroxymethyluracil in their DNA to select those that are defective in DNA synthesis under restrictive conditions. The pyrimidine, (14)C-hydroxymethyluracil (HMU), is not incorporated into bacterial or phage DNA. Neither HMU nor dHMU can replace thymine as a growth requirement for Bacillus subtilis 168 Ind(-) Thy(-). HMU does not inhibit the utilization of thymine. Although dHMU inhibits deoxythymidine utilization, the inhibition is not competitive.     

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.     

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E. coli DNA until 6-7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.     

Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae.

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.     

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.     

Influence of Bacteriophage PBS1 and φW-14 Deoxyribonucleic Acids on Homologous Deoxyribonucleic Acid Uptake and Transformation in Competent Bacillus subtilis

Both bacteriophage PBS1 deoxyribonucleic acid (DNA) (in which all the thymine residues are replaced by uracil) and phage W-14 DNA [in which half the thymine residues are replaced by 5-(aminobutylaminomethyl)uracil or 5-putrescinylthymine] exhibit comparable competing abilities for uptake of homologous DNA in a Bacillus subtilis competent system. But, whereas PBS1 DNA leads to a decrease in transformation frequencies compatible with its competing ability for DNA uptake, W-14 DNA decreases transformation frequencies by a factor up to eightfold higher. The effect of W-14 DNA on transformation frequencies is visible even at a concentration level that does not decrease transforming DNA uptake. No such effect was observed with heterologous DNA containing presumably ionically bound putrescine. Low concentrations of W-14 DNA decreased the number of double (nonlinked) transformants more than single transformants. The influence on transformation was abolished when W-14 DNA was added 20 min after addition of transforming DNA, i.e., when the recombination process was terminated. The putrescine-containing DNA also decreased retention of trichloroacetic acid-precipitable radioactivity of homologous DNA taken up. We conclude that W-14 DNA inhibits some intracellular process(es) at the level of recombination. In addition, there is evidence that W-14 DNA, but not heterologous DNA with ionically bound putrescine, binds also to site(s) on the cell surface other than receptors for homologous DNA.     

Host-Mediated Repair of Discontinuities in DNA From T4 Bacteriophage

Discontinuities of T4 DNA which are caused by excision of UV-damaged areas, by decay of (32)P atoms, or which are present in DNA from rII(-)lig(am) (-) phage produced in a host nonpermissive for amber mutants are all repaired by bacterial enzymes after infection in the presence of chloramphenicol. Escherichia coli DNA polymerase I participates in the host-mediated repair, but an approximately 20-fold variation in the levels of host polynucleotide ligase does not affect either the kinetics or the extent of repair observed. Upon removal of chloramphenicol, host-repaired DNA from UV-irradiated phage undergoes a secondary cycle of breakage, which ultimately results in solubilization of most of the phage DNA. If the cells are co-infected with nonirradiated helper phage, the secondary breaks are repaired and the continuity of the polynucleotide chain is restored. The close coincidence in the extent of primary and secondary breakage suggests that phage-coded enzymes recognize and excise areas improperly repaired by the host. In contrast to host-mediated repair, repair mediated by rescuing phage probably restored functionality to the damaged DNA.     

Selection and Preliminary Characterization of Temperature-Sensitive Mutants of Type 5 Adenovirus

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wild-type strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1%. All eight mutants had reversion frequencies of 10(-5) or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3- to a 3,000-fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25% of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).     

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.     

Abortive replication of choleraphage phi 149 in Vibrio cholerae biotype el tor.

Choleraphage phi 149 adsorbed irreversibly to Vibrio cholerae biotype el tor cells, and 50% of the injected phage DNA bound to the cell membrane. Although no infectious centers were produced at any time during infection, the host macromolecular syntheses were shut off and the host DNA underwent chloramphenicol-inhibitable degradation. Synthesis of monomeric phage DNA continued similar to that observed in the permissive host. However, the concatemeric DNA intermediates produced were unstable and could not be chased to mature phage DNA. Pulse-labeling of UV-irradiated infected cells at different times during infection allowed identification of phage-specific proteins made in this nonpermissive host. Although most of the early proteins were made, only some of the late proteins were transiently synthesized.     

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

Normal levels of the hypermodified pyrimidine, alpha-putrescinylthymine, which is formed from hydhydroxymethyluracil at the polynucleotide level (Maltman et al., J. Virol. 34:354-359, 1984), are not required in bacteriophage luminal diameterW-14 DNA for the DNA to serve as a replicative template in luminal diameterW-14-infected cells.     

Polymer-level synthesis of oxopyrimidine deoxynucleotides by Bacillus subtilis phage SP10: characterization of modification-defective mutants.

Bacillus subtilis phage SP10 DNA has two oxopyrimidines, thymidine 5'-monophosphate (dTMP) and its hypermodified analog (YdTMP). Published data suggest that both are synthesized by postreplicational modification of 5-hydroxymethyldeoxyuridylate (HOMedUMP) in nascent DNA by the following pathway: HOMedUMP----PPOMedUMP----dTMP (85%) or YdTMP (15%); PPOMedUMP is 5-(hydroxymethyl-O-pyrophosphoryl)deoxyuridylate, the pyrophosphoric acid ester of the C5CH2OH function of HOMedUMP. This paper describes aberrant DNAs synthesized at nonpermissive temperatures by a complementary series of heat-sensitive, modification-defective (mod) mutants. Collectively, these mutants encompass the major steps in the complete modification of nascent SP10 DNA. DNA produced by modA phage retains HOMedUMP as its sole oxopyrimidine, implying that (i) this mutant is defective in the pyrophosphorylation step and (ii) formation of PPOMedUMP is required for any further modification. Furthermore, studies with double mutants indicated that modA is epistatic for all other mod mutants, which supports the hypothesis that modA controls the earliest step in the modification pathway. Since their DNAs contain no YdTMP, modC and modD are defective in hypermodification (i.e., PPOMedUMP----YdTMP). However, dTMP occupies the entire oxopyrimidine fraction of modC DNA, whereas modD DNA has a normal dTMP content, but the now-missing YdTMP is replaced by either PPOMedUMP or a byproduct of abortive hypermodification. It is proposed that the modD mutants are defective in the catalytic aspects of hypermodification and that modC are defective in some regulatory function that promotes hypermodification at the expense of reductive modification (i.e., PPOMedUMP----dTMP). Reductive modification is defective in modB phage, as evidenced by the absence of dTMP. In contrast to the others, modB DNA has a complex oxopyrimidine content: HOMedUMP, ca. 30%; PPOMedUMP, ca. 40%; and YdTMP, ca. 30%. The expanded level of YdTMP suggests that at certain sites, reductive modification and hypermodification are competing reactions. Interestingly, the PPOMedUMP content of modB DNA seemingly reflects the maximum degree to which phage DNA can be pyrophosphorylated, since the loss of YdTMP from modBmodC and modBmodD DNAs results in a unilateral increase in HOMedUMP content.     

Effect of Elevated Temperature on Deoxyribonucleic Acid Synthesis in Bacteriophage φ29-Infected Bacillus amyloliquefaciens

Deoxyribonucleic acid (DNA) synthesis in bacteriophage phi29-infected Bacillus amyloliquefaciens was studied at 37 and 45 C. Infectious intracellular particles appear at the same time at both temperatures, but the average burst size is reduced 45 to 50% at 45 C. There is a transient inhibition of cellular mass increase at 45 C which is not observed at the lower temperature. In addition, the rate of host DNA synthesis is reduced and the onset of viral-specific DNA replication is delayed for 6 to 9 min at 45 C. These findings allowed us to screen phage phi29 mutants which are sensitive to growth at 45 C for their ability to synthesize phi29 DNA in the absence of host DNA replication. We obtained mutants which make no viral DNA, reduced levels of DNA, or normal quantities of DNA under nonpermissive conditions. Pulse-labeled viral DNA which sediments more rapidly than mature phi29 DNA molecules was observed after gentle cell lysis and zone sedimentation. This DNA is not a precursor of normally sedimenting phi29 DNA and apparently consists of mature phi29 DNA molecules aggregated with large pieces of bacterial DNA.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.