Über die Mehrfachreaktivierung des Infektions- und Lysogenisierungsvermögens des Phagen P22 von Salmonella typhimurium nach UV-Bestrahlung

Summary It has been shown, that uv-lesions in the DNA of the temperate Salmonella bacteriophage P22 can undergo multiplicity reactivation. The multiplicity reactivation is very efficient and does not seem to be hindered by recombination events between phage and host genetic material as it is the case with the Coli phage T1. The ability of P22 to lysogenize its host cell is very sensitive to low doses of UV; its inactivation by UV can also undergo multiplicity reactivation.     

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.     

Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation.

A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA. (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates. A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.     

Multiplicity reactivation and repair of nitrous acid-induced lesions in bacteriophage T4

Nitrous acid-induced lesions in phage T4 were shown to be efficiently repaired by multiplicity reactivation. Mutants defective in genes 32, 46, 47, x and y showed substantially less MR ² of these lesions than wild type. The gene 47 mutant, which showed the least MR of nitrous acid lesions, also showed virtually no MR of ultraviolet lesions. Mutations in genes 30, 44 and v did not affect MR of nitrous acid-induced lesions. Each of the mutations which lowered MR was shown previously to reduce recombination. Our results suggest that the gene functions employed in MR are the same functions used in genetic recombination, and, based on this, we propose a tentative recombinational model for MR.The mutants defective in genes 32, 46 and 47, which are deficient in recombination, were shown to be more sensitive to nitrous acid inactivation than wild-type phage upon single infection. Our results indicate that, in wild-type single infections, about 20% of nitrous acid-induced lethal lesions may be repaired by a recombinational post-replication form of repair.     

Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Analyses of spontaneous mutations of cloned gene 49 of phage T4

Holliday structure resolving enzyme endonuclease VII (endo VII) of phage T4 is highly toxic for E. coli when expressed outside of the phage infection environment. As a consequence, plasmids with a mutated gene 49, the gene which encodes for endo VII, can be easily isolated and characterised. We have isolated and characterised 400 survivors from independent transformations with a plasmid carrying gene 49 under the control of the T7 promoter. The majority had mutated gene 49 by IS10 insertions which almost exclusively mapped to a distinct site. When this site was mutated other insertion sites were observed as well as an increase in other mutational events including large deletions. Neither of the observed insertion sites mapped matched the consensus IS10 sequence completely. Additionally when the level of expression of gene 49 was altered the distribution of mutations was changed suggesting that other elements apart from the target sequence are necessary for determining IS10 insertion. The expression of gene 49 in E. coli provides a particularly useful tool for the analysis of mutational events.     

On the lack of host-cell reactivation of UV-irradiated phage T5. I. Interference of T5 infection with the host-cell reactivation of phage T1

UV-irradiated phage T5, in contrast to T1, T3 and T7, fail to display hostcell reactivation (HCR) when infecting excision-repair proficient Escherichia coli cells. Possible causes of this lack of HCR (which T5 shares with the T-even phages) have been investigated by studying HCR of T1 under conditions of superinfection by T5. Repair-proficient B/r cells were infected at low multiplicity with UV-irradiated phage T1 in the presence of 1.8 mg/ml caffeine and were superinfected after 15 min with heavily UV-irradiated T5 amber mutants at high multiplicity. The caffeine, which is later diluted out, prevents any T1 repair prior to T5 superinfection, and UV (254 nm) irradiation of T5 with 144 J/m² reduces the ability of this phage to exclude T1, thus permitting a reasonable fraction of the mixedly infected complexes to produce T1 progeny.Under these conditions, T5 superinfection causes loss of HCR in about 90% of the T1-producing complexes. Superinfection with unirradiated T5 likewise inhibits HCR of T1, but superinfection with irradiated T3 (a host-cell-reactivable phage) does not. This indicates that the observed HCR inhibition of T1 results from T5 infection rather than from competition of irradiated foreign DNA for the excision-repair enzymes of the bacterial host. Employment of apropriate T5 amber mutants has shown that “first-step transfer” (FST) of T5 DNA (involving only 8% of the T5 genome) is sufficient for HCR inhibition, but that transfer of the remainder DNA in addition inhibits a previously described minor T1 recovery process. HCR inhibition of T1, and thus presumably lack of HCR in T5 itself, is ascribed to a substance which is produced either post infection by a gene located in the FST segment of the T5 genome, or which is transferred from extracellular T5 together with the FST DNA.     

On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5.The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the “second-step-transfer” region of T5 DNA (involving the remainder of the genome). Superinfection with T4ν₁ phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

On the repair mechanism responsible for multiplicity reactivation in bacteriophage T4

Summary Multiplicity reactivation has been studied using two T4 phages carrying a large number, twenty six, of genetic markers covering the whole viral genome. It has been found that in the progeny of irradiated phages the observed increase of recombination frequencies is not limited to the vicinity of radiation-induced lethal lesions, it can occur all along the phage genome. Most of the single bursts produced by multiplicity reactivation contain phages of entirely parental genotype. This fact may reveal the existence of repair by recombination between replication products of a single viral genome.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

A T7 amber mutant defective in DNA-Binding protein

Summary A T7 amber mutant, UP-2, in the gene for T7 DNA-binding protein was isolated from mutants that could not grow on sup ⁺ ssb-1 bacteria but could grow on glnU ssb-1 and sup ⁺ ssb ⁺bacteria. The mutant phage synthesized a smaller amber polypeptide (28,000 daltons) than T7 wild-type DNA-dinding protein (32,000 daltons). DNA synthesis of the UP-2 mutant in sup ⁺ ssb-1 cells was severely inhibited and the first round of replication was found to be repressed. The abilities for genetic recombination and DNA repair were also low even in permissive hosts compared with those of wild-type phage. Moreover, recombination intermediate T7 DNA molecules were not formed in UP-2 infected nonpermissive cells. The gene that codes for DNA-binding protein is referred to as gene 2.5 since the mutation was mapped between gene 2 and gene 3.     

Vergleichende Untersuchungen an HNO2-Inaktivierten und UV-Inaktivierten Bakteriophagen T4

Summary The inactivation of phage T4 by nitrous acid (HNO₂) is essentially an exponential function of time of treatment. HNO₂-inactivated T4 is able to undergo multiplicity reactivation, and genetic markers may be rescued by live phage, however, the extent of both effects is appreciably less than after UV-inactivation. Also, the survival of phenotypic function of the cistronsr II-A andr II-B is lower with HNO₂-treatment than with a UV-irradiation of a corresponding number of hits.The reduced effects are quantitatively accounted for by the assumption of lethal hits blocking early steps of infection. These early-step damages amount to approximately 1/6 of the total hit number; it is still unknown whether they occur in DNA or in protein. Some indication for the occurrence in protein comes from the result that the host-killing efficiency of HNO₂-inactivated phage is reduced at a similar rate as these early-step damages occur. However, at least 5/6 of the lethal hits are due to chemical changes within the DNA, as can be calculated from the results of multiplicity reactivation, marker rescue, and phenotypic survival of therII-cistrons.

---

---

Mit 6 Textabbildungen     

Participation of bacteriophage T4 gene 41 in replication repair

The location of the non-essential T4 mutant uvs79, with defective replication repair, is described. After crosses with double mutants dispersed over the early region of T4, a linkage was observed with the double mutant am41 : am42. For more accurate location, crosses were made with single mutants. Uvs79 proved to be located between mutants amC23 and amN81 in gene 41, as shown by 3-point crosses. No genetic complementation with respect to multiplicity reactivation was found between amN81 and uvs79 after a co-infection of an su^? host. Apparently, mutant amN81 is disturbed as to replication repair and, owing to its lack of DNA synthesis, also in replication-dependent recombination repair. Consequently, the product of gene 41 has a function additional to its RNA-primer induction during replication of undamaged DNA. Presumably, the product of gene 41 induces RNA primers opposite DNA regions containing lesions. This capability is believed to be specifically affected by the uvs79 mutation.     

A mutation in Escherichia coli enhancing the UV-mutability of phage lambda but not of its infectious DNA in a spheroplast assay

Summary A mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage . The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage . However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated -DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.The mutation responsible for the mul phenotype has been mapped by conjugation and transduction to be located between the markers pyrE and ilv and probably marks a new gene. The mul mutation does not increase the UV-sensitivity of the cells nor does it affect their ability to perform host cell reactivation. The presence of a recA allel in a mul mutant abolishes the UV-mutability of phage .Part of this work is from the thesis of the first author in partial fullfilment of the requirements for the doctoral degree of the University of Bochum. Some of the results have already been presented at the 32. Kongreß für Hygiene und Mikrobiologie at Münster, 1969.     

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvsY) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication. Received: 3 February 1999 / Accepted: 20 July 1999