Weigle reactivation of the single-stranded DNA phage f1

Summary The survival of ultraviolet light (UV) damaged single-stranded DNA bacteriophage f1 is increased when the Escherichia coli host is irradiated with UV prior to infection. This repair, called Weigle reactivation, is multiplicity independent and is absent in recA and in lexA mutants. The function of the recA-lexA repair system needed is repair and not recombination, as demonstrated by the absence of Weigle reactivation in mutants that are recombination proficient but defective in repair of double-stranded DNA. Weigle reactivation of f1 requires high levels of the recA protein, and in addition activation of recA or another protein. This activation can be produced by UV irradiation, or by the tif-1 allele of recA together with the spr allele of lexA. Mutagenesis of f1 has the same requirements as W-reactivation, and in addition requires UV irradiation of the phage.     

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

Summary The ability to reactivate ultraviolet (UV) damaged phage CbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage CbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2–2 h at 30°C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5–10 J/m² of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.     

Relation between UV suppression of polarity in phi X174 and UV sensitivity of rho mutants.

The suppression of polarity by UV irradiation was similar to the suppression by rho mutants. This was demonstrated for a polar nonsense mutant of phage phi X174. Treatment of the host for 30 min with 100 micrograms of the radiomimetic drug mitomycin C per ml was about as effective as 550 J of UV irradiation per m2 in relieving polarity. The shape of the UV survival curves for rho mutants could be linked to a proposed mechanism of UV relief of polarity. Host cell reactivation of phage lambda and W-reactivation of phage G4 were unaffected by rho mutations. UV suppression of polarity is independent of the Hcr- and RecA- phenotypes. An explanation for the UV sensitivity of rho mutants is provided, and several ways are considered in which UV irradiation may deplete cellular rho activity and thereby cause UV relief of polarity. We propose a novel theory that relates the UV inactivation of normal repair-proficient cells to a decrease in rho activity.     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Participation of rec genes of Escherichia coli K 12 in W-reactivation of UV-irradiated phage lambda.

The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored. In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment. Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA]. W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation.     

W-reactivation and W-mutagenesis of gamma-irradiated phage lambda.

UV irradiation of Escherichia coli wild-type cells manifested the phenomena of W-reactivation (WR) and W-mutagenesis (WM) of phage lambda irradiated by 60Co gamma-rays in broth. WR of gamma-irradiated phage was half as efficient as that of UV-irradiated phage, although the frequency of c mutations in conditions of WR was about the same in both phages. The xthA and recBrecC sbcB mutants were practically identical with wild-type cells in respect of WR and WM of UV- and gamma-irradiated phage. As in UV-irradiated phage, WR and WM of gamma-irradiated phage were absolutely dependent on the recA+ and lexA+ genes of the host cell. WR and WM required much smaller doses of UV radiation for induction in polA1 and uvrB mutants. The lig-ts mutant, temperature sensitive in polynucleotide ligase, was deficient in WR and WM of UV- and gamma-irradiated phage at the semi-permissive temperature of 37 degrees. The uvrE502 mutant and the allelic recL152 strain were absolutely deficient in WR and WM of gamma-irradiated phage. In UV-irradiated phage WR was reduced, but not eliminated, in the uvrE mutant, and WM was entirely suppressed. This is another example of uncoupling of WR and WM which shows that several repair systems are active in WR but only some of them are mutagenic.     

UV-induced reactivation and mutagenesis ofλ-phages after treatment with 8-Methoxypsoralen or thiopyronine and light

Summary Lesions, which were produced on-phages DNA by the photosensitization effect of 8-methoxypsoralen (8-MOP) can be repaired by UV-induced repair systems (W-reactivation) inEscherichia coli wild type host cells. By optimum induction of the repair system, about 45% of the 8-MOP lesions are repaired. The survival of-phages inactivated by the photodynamic action of thiopyronine (TP) is only negligibly increased by the same UV-induced repair systems. However, the frequencies of clear plaque mutations of 8-MOP treated as well as TP treated-phages increase in similar fashion if the host cells of wild type have been irradiated with UV. These results show the different capacities of induced repair systems in repairing different types of lesions. They also suggest that some types of base damages are repaired more error-prone than other DNA-lesions.     

Inducible, error-free DNA repair in tsl recA mutants of E. coli

Summary Host cell reactivation and UV reactivation and mutagenesis of UV-irradiated phage were measured in tsl recA ⁺ and tsl recA host mutants. Host cell reactivation was slightly more efficient in the tsl recA strain compared to the tsl ⁺ recA strain. Phage was UV-reactivated in the tsl recA strain with about one-half the efficiency of that in the wild type strain, but there was no corresponding mutagenesis of phage. UV-reactivation was also slightly lower and mutagenesis several-fold lower than normal in the tsl recA ⁺ strain. To account for these observations, we propose that there is an inducible, error-free pathway of DNA repair in E. coli that competes with error-prone repair for repair of phage lesions.     

W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Heat shock stress in Bacteroides fragilis

The response to heat shock was investigated in the obligate anaerobe Bacteroides fragilis. The cells responded quickly to stress and synthesised seven heat shock proteins immediately upon exposure to heat. The apparent molecular weights of the seven proteins differed from the apparent molecular weights of the proteins induced by UV irradiation, O₂ and H₂O₂. Heat shock did not induce phage reactivation whereas UV irradiation, O₂ and H₂O₂ did induce phage reactivation systems. Ethanol did not elicit the heat shock response. Two heat resistant B. fragilis mutants were isolated. Both mutants lost the ability to synthesise the same two heat shock proteins. It is concluded that the heat shock response and the responses to UV irradiation, O₂ and H₂O₂ represent two independent groups of stress responses in B. fragilis.     

Induced reactivity of UV-damaged phage gamma in E. coli K12 host cells treated with aflatoxin B1 metabolites.

The metabolites of aflatoxin B1, the most potent hepatocarcinogen so far known, promote in E. coli K12 cells the reactivation of phage lambda damaged by ultraviolet (UV) radiation. This reactivation process is error prone; 25% of the phage DNA lesions are repaired, but mutagenesis, scored as clear plaque formation, is increased as much as 10-fold. Such reactivation of UV-damaged phage lambda, which occurs in wild-type and in uvrA but not in recA bacteria, is inducible: phage reactivation is obtained even after a long delay following treatment of the host by the short-lived metabolites. This induced reactivation of UV-damaged phage in hosts treated with metabolites of aflatoxin B1 is similar to direct of indirect UV reactivation. Metabolites of aflatoxin B1 produce induced phage reactivation as well as prophage lambda induction in lysogens and cell filamentation in non-lysogens. These cellular events are also triggered by DNA lesions caused by UV radiation and result from the induction of a metabolic pathway (SOS functions). We postulate that, in eucaryotes, carcinogens may induce cellular SOS functions similar to those in E. coli. Induction of such functions might be responsible for the transformation of mammalian cells.     

Weigle reactivation of diploid M13 phage

Summary The limited ability of ultraviolet (UV)-irradiated E. coli cells to W-reactivate UV-irradiated, single-stranded DNA phages fd and M13 was investigated. The kinetics of induction for W-reactivation of UV-irradiated fd phage are different from that for other SOS functions. W-reactivation of UV-irradiated M13 phage was studied using phage particles that contain at least two single-stranded DNA genomes. No effect on the extent of W-reactivation of diploid phage was observed, compared to that of normal haploid phage, indicating that the mechanism of W-reactivation of single-stranded DNA phages does not involve recombination between partially replicated genomes.     

W-reactivation and W-mutagenesis in UV-irradiated phage phil05 of Bacillus subtilis]

The survival of UV-irradiated phage ?105 on the lawns of several strains of Bacillus subtilis: wild type (strain 168) and 11 recombination-defficient mutants (recA1, recB2, recB3, recB19, recD27, recF15, recF18, recK4, recM13, recL16 and recO61) was investigated. All rec mutants have the phenotype Hcr+, i.e. normally host-cell reactivate UV-damaged phage. Small doses of UV-irradiation given to the wild type (rec+) cells increase the probability of survival of UV-irradiated ?105 phage (W-reactivation) and significantly enhance the frequency of c-mutants (W-mutagenesis). Maximal frequency of clear mutations in conditions of W-mutagenesis is 3-10(-3), i.e. is 100 times higher than the spontaneous background. Various rec mutations of host cells only diminish the level of W-reactivation but do not eliminate it completely. The most deficient in W-reactivation is recD27 mutant. Mutations recB2, B3, B19 and O61 have no effect on W-mutagenesis of UV-irradiated phage ?105 and on UV-induction of ?105, F15,F18 and L16 mutants. UV-irradiation of lysogenic cells of these mutants does not induce ?105 prophage.     

Effects of x irradiation on a temperate bacteriophage of Haemophilus influenzae.

The inactivation of bacteriophage HP1c1 by X rays in a complex medium was found to be exponential, with a D0 (the X-ray exposure necessary to reduce the survival of the phage to 37%) of approximately 90 kR. Analysis of results of sucrose sedimentation of DNA from X-irradiated whole phage showed that the D0 for intactness of single strands was about 105kR, and for intactness of double strands, it was much higher. The D0 for attachment of X-irradiated phage to the host was roughly estimated as about 1,100 kR. Loss of DNA from the phage occurred and was probably due to lysis of the phage by X irradiation, but the significance of the damage is not clear. The production of single-strand breaks approaches the rate of survival loss after X irradiation. However, single-strand breaks produced by UV irradiation, in the presence of H2O2, equivalent to 215 kR of X rays, showed no lethal effect on the phage. Although UV-sensitive mutants of the host cell, Haemophilus influenzae, have been shown to reactivate UV-irradiated phage less than does the wild-type host cell, X-irradiated phage survive equally well on the mutants as on the wild type, a fact suggesting that other repair systems are involved in X-ray repair.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Peroxide inducible phage reactivation in Bacteroides fragilis

Abstract Reactivation of UV-irradiated phage b-1 was induced by H₂O₂ and UV in Bacteroides fragilis . The characteristics of H₂O₂ and UV induced phage reactivation differ from a previously reported oxygen induced reactivation system. The survival of B. fragilis cells after UV irradiation was also increased by pretreatment with H₂O₂. DNA synthesis was not inhibited in the host cells exposed to H₂O₂ concentrations which induced phage reactivation. The pattern of DNA degradation and synthesis after UV irradiation with and without H₂O₂ differed from the effect of O₂ on DNA synthesis in irradiated B. fragilis cells.     

Reactivation and mutagenesis of UV irradiated lambda phage in bacteria treated with platinum (II) compounds

Treatment of wild type Escherichia coli with cis -Pt(NH3)2Cl2 increased the survival and frequency of clear plaques formation of lambda phage damaged by UV radiation. The reactivation process was present in an uvrA mutant and abolished in a lexA host. Trans-Pt(NH3)2Cl2 and [Pt(dien) Cl]Cl (dien = 2HN-CH2-CH2NH-CH2-CH2-NH2) which, inhibited DNA synthesis less than the cis isomer or not at all, respectively, induced only a slight increase in survival of UV irradiated phage while mutagenesis was not affected. A relation exists between the reactivation of UV damaged phage in bacteria treated with these three compounds and their recently reported abilities to inhibit DNA synthesis and induce recA protein.     

Studies on radiation-sensitive mutants of E. coli. II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda

Summary It has been shown that linear DNA molecules of phage are converted to the twisted circular structure (species I) by covalent closure of the both strands at the cohesive ends after infection to the immune bacteria and that the twisted circular molecules are transformed to the circular form (species II) by a single-strand break in one of the strands of their DNA. This system offers a very sensitive method to study on the strand breaks or their repair. For characterization of the defects of ultraviolet sensitive strains, the structural changes of ultraviolet irradiated DNA in these strains were studied.Ultraviolet irradiation to phage greatly reduced the extent of conversion of the molecules to the species I in the uvrD mutant while the irradiation showed little effect on the conversion in the uvrA, B and C mutants. When infected bacteria carrying species I molecules were irradiated, the species I molecules in the uvrD mutant were disrupted while most of the molecules in the uvrA, B and C mutants kept the structure. These results indicate that in the irradiated DNA strand breaks are rarely introduced or, if introduced, repaired rapidly in the uvrA, B and C mutants and they are introduced in the uvrD mutant leading to the degradation of the DNA. These results provide a firm evidence that the defect of the uvrD mutant is different from other Her^- mutants and in the process of repair synthesis.Ultraviolet irradiation to the uvrD mutants promote the formation of the species I molecules from the infected irradiated -DNA.Such effect was not observed with the uvrA mutant. Since the uvrD mutant has UV reactivation capacity and the uvrA mutant has not, the above phenomenon is probably caused by UV reactivation and may provide a more direct method to study the mechanisms of UV reactivation than the plaque assay.Abbreviations used UV Ultraviolet light - UVr Ultraviolet light reactivation This work was aided in part by a research grant GM 08384 from the United States Public Health Service.