Expression of an excision repair gene in transformation of Bacillus subtilis.

Summary DNA of Bacillus subtilis proficient in excision repair (hcr ⁺) was introduced into Angiografinpurified competent cells of an excision repair-deficient strain UVS-1 (hcr-1). The hcr ⁺ gene was found to affect the UV-survival curve of the cells, giving rise to a UV-resistant component. However, a considerable number of colonies of the UV-resistant component consisted of cells that were not transformed to hcr ⁺ as judged by their sensitivity to mitomycin C (MC), UV, and by their ability to reactivate UV-irradiated M2 phages. This suggests that the hcr gene may be expressed without integration. The recA function of B. subtilis was necessary for expression of UV resistance to occur. When DNA-treated cells were selected for met ⁺ recombinants, the UV-resistant component was again found on the UV-survival curve and about half of the colonies of the UV-resistant component consisted of Hcr^- cells. This result was explained by an integration-segregation model for hcr ⁺ and met ⁺ genes. The effect of the hcr ⁺ gene was seen even when DNA was added after cells were irradiated with UV, although this effect was gradually diminished by delaying the time of DNA addition. A complementation effect was found between two excision repair mutations residing in two distant loci, using hcr-114 DNA as a donor and hcr-1 cells as a recipient.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Reactivation of photodynamically inactivated lambda phages

Summary UV-irradiated c phages show a lower survival when plated on rec ^–-cells as compared to rec ⁺-cells. Photodynamically inactivated c phages show the lowest survival on hcr ^–; rec ^–-cells. The rec-functions do not influence the repair of UV- or photodynamically induced lesions in T1 phages.     

UV-sensitivity and UV-mutability of infectious lambdaDNA: reactivation, protection and mutability in various assay systems

Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca⁺⁺-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr^- spheroplasts and cells, resp., and since survival is also identical in Ca⁺⁺-treated Hcr⁺ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr⁺ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca⁺⁺-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca⁺⁺-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul ^-, could not be induced in DNA when assayed with spheroplasts or Ca⁺⁺-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.     

Mutagenicity of reactive derivatives of carcinogenic hydrocarbons: evidence of DNA repair

The ability of cellular DNA repair enzymes, which are active on ultraviolet light-induced lesions in DNA, to recognize and repair damage induced in DNA by exposure to carcinogenic polycylic hydrocarbons was investigated and the effect of such repair processes on the mutagenicity of the hydrocarbons determined. The carcinogenic hydrocarbos, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA), chosen for this study because they form well characterized, stable products with DNA, were dissolved at various concentrations in acetone, added under mild conditions to biologically active DNA isolated from Bacillus subtilis, and the reaction stopped by ethanol precipitation. The hydrocarbons were determined by specific radioactivity to be covalently linked to DNA at a frequency of from 1–5 per 1000 nucleotides. An increased frequency of bound hydrocarbon molecules was directly correlated with a decrease in the buoyant density of the DNA as measured in analytical CsCl centrifugation studies. The samples of hydrocarbon-bound DNA were tested for survival of biological activity and for the frequency of induced forward mutations in two recipient strains (hcr⁺ and hcr^?) of Bacillus subtilis which differ in their ability to repair ultraviolet light-induced lesions in DNA. The survival of the biological activity was significantly higher in the repairing strain (hcr⁺). A higher frequency of mutations was detected in the repairing strain as well. The loss of transforming activity and the increase in the frequency of mutations (up to 20-fold) was directly proportional to the amount of hydrocarbon bound to the DNA samples. The majority of these mutations proved unable to revert spontaneously. Finally, the ability of highly purified rat liver endonuclease, shown to recognize lesions in UV-irradiated DNA, to recognize such hydrocarbon lesions was investigated. Tritiated 7-BrMeBA-treated DNAs exposed to the enzyme were found to sustain single-strand nicks in proportion to the amount of hydrocarbon bound while untreated DNA remained substantially intact. The action of the endonuclease appeared to result in an increase in the biological activity of DNA containing hydrocarbon residues when this was assayed in the hcr^? mutant.     

Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs

A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented. Phages phi 29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated. For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr+ host. The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production. Even though phi 29 has the smallest target size of the three phages, phi 29 and its DNA are the most sensitive. Phages SPO2 and SPP1 code for gene products which complement the repair system of the host. The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr-host. This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur. The recombination process strongly interferes with the repair of damaged DNA.     

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.     

Restriction and modification in B. subtilis

Summary The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated.The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted.In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands¹ alone, is significantly lower than that of native DNA.The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.Abbreviations EGTA ethyleneglycolbis-(aminoethylether)tetraacetic acid - m⁺ modified - m^- non-modified - moi multiplicity of infection - r⁺ m⁺ restricting and modifying - r^- m^- mon-restricting and non-modifying - SSC 0.15M NaCl+0.015 M trisodium citrate - SDS sodium dodecyl sulphate     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

Der Einfluss extrazellul?rer UV-Bestrahlung des Phagen T2 auf die Replikation Seiner DNA

Summary The effects of extracellular UV-irradiation on the replication of DNA were tested with phage T2. Cells ofE. coli B/1 were multiply infected with UV-irradiated T2. The kinetics of P³²-incorporation into phage DNA were significantly different from the unirradiated control.With unirradiated phages the time curve is linear during the second half of the latent period after as short nonlinear increase. With UV-irradiated phages however the amount of DNA increases exponentially during the whole latent period. Such kinetics might be expected from semiconservative DNA-replication if templates were limiting. The reported findings suggest that other regulatory mechanisms normally limiting DNA-synthesis are inactivated by UV. The kinetics determined by semiconservative replication could then be clearly observed with irradiated phages. The nature of the normally regulating mechanisms is discussed.

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Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Differences in host controlled reactivation of lambdoid and T phages

Summary Phages 434, T4, T5 and T7 are studied with regard to host controlled reactivation of damage produced by UV or photodynamic action sensitized by thiopyronine. Repair of 434 phages proceeds under control of both hcr and rec genes. UV irradiated T5 and T7 phages are reactivated under control of the host's hcr genes only. If these phages are inactivated by photodynamic action they are reactivated not at all. T4 phages inactivated by both treatments are also refractory to host controlled reactivation. These differences might reflect different degrees of autarchy and different abilities of phage DNAs to serve as substrate for recombination enzymes of the host.These results were presented in an abstracted version at the V. International UV colloquium Grundlagen der UV-Wirkung, Kühlungsborn, DDR, in October 1969. The experiments with T5 were done by Mrs. E. Marx.     

Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F⁺, hcr⁻, thy⁻) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with ³H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

The enhancement of the mutagenic effect of ultraviolet radiation inEscherichia coli by caffeine and acriflavine

The mutational synergism of caffeine and acriflavine was studied in five types ofEscherichia coli mutants induced by u. v.-radiation. The following types of mutations were compared: streptomycinrresistance (strain B/r), streptomycin-independence (strain Sd-4), and reversions to prototrophy (strains WP-14 pro⁻, WP-2 try⁻, and WP-2 try⁻ hcr⁻). In all hcr⁺ strains tested the presence of caffeine or acriflavine in a post-irradiation plate medium slightly decreases the survival of u.v.-irradiated cells and increases considerably the frequency of induced mutations. The mutational synergism of caffeine and acriflavine in the str-r and str-i mutants is observed only within the range of low doses. The abovementioned dose-dependence of the synergistic effect is discussed from the point of view of qualitative difference between the premutational damage caused by low and high doses. The post-irradiation treatment by caffeine slightly increases the frequency of induced prototrophs also in the WP-2 hcr⁻ strain. This finding is explained by the inhibition of the residual HCR-activity of the strain. The post-irradiation mutational synergism of acriflavine was not found in the WP-2 hcr⁻ strain.     

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).     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA⁺ or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD⁺ allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.     

Induction of c-mutations in extracellular phage lambda by gamma-rays

Summary Mutagenic action of ⁶⁰Co -rays on extracellular phages red ⁺ and red1 13 after irradiation in 4% nutrient broth in the absence or in the presence of 0.1 M cysteamine or in dried samples was studied. The yield of c mutations was almost independent of the repair genotype of the host cells (uvrA6, polA1, recA13, lexA102, uvrE502, uvrD3 or xthA9), of the phage Red function and of the conditions of -irradiation and was 1·10^-12 per base pair and 1 rad. When the SOS-repair system of the host cells was induced by moderate UV irradiation, the yield of c-mutations was drastically enhanced in phage irradiated in broth, but not in phage irradiated in the dried state. These data allow us to suppose that the direct action of -rays induces, in phage DNA, premutational lesions that are fixed into mutations by replication. On the other hand after -irradiation in broth, when indirect radiation effects are only partially suppressed, about 85% of premutational lesions are converted into mutations by means of the inducible, errorprone SOS-repair system.     

Effect of bacterial host repair systems on the viability of hydroxylamine and methyl methanesulfonate treated T4 and lambda bacteriophages

Summary Survival of HA or MMS-treated T4 and lambda phages was estimated in bacterial cells differing in their ability to repair DNA. It has been found that the mismatch repair system of the bacterial host, which involvesmutSmutRmutLuvrE anddam loci, does not excise, or does so to only a limited extent, the nonpaired bases from DNA of HA or MMS-treated phages. Mutation inpolA, both in the polymerase as well as in the 53 exonuclease activity, have a small effect on survival of HA-treated phages, whereas mutation in the polymerase activity has a pronounced effect on survival of MMS-treated phages. There was a difference in the effect of polA mutations on survival of MMS-treated T4 and lambda phages; the survival of the former was less affected than the latter. Induction of SOS response has no effect on repair of HA and MMS-treated phages. Pretreatment of bacterial host (including theada ^- mutant) with low doses of alkylating agents increases the survival of MMS (but not HA)-treated phages; pretreatment of bacteria with HA has no effect on survival of HA-treated phages. Three lines of evidence: the different inactivation rates of MMS-treated T4 and lambda phages, variation in the effect ofpolA mutations on survival of T4 and lambda phages, and a different level of adaptive response inada ^- cells towards of MMS-treated T4 and lambda phages, suggest that the patterns of DNA methylation in T4 and lambda phages are different.     

Infection of transformable cells ofHaemophilus influenzae by bacteriophage and bacteriophage DNA

Summary A phage HP1, infecting transformable cells ofHaemophilus influenzae Rd, has been isolated. The general properties of the wild type and of a clear plaquemutantc1 employed for most of the experiments are described. Phage DNA is infective for transformableHaemophilus cells with an efficiency (plaqueforming units of the original phage recovered as DNA-infected cells) of up to 6×10^–3. The competence ofHaemophilus cells for infection with phage DNA parallels the competence for transformation with bacterial DNA.Both HP1 and thec1 mutant are able to lysogenize their host, and the lysogenic cells are readily induced by UV. Competent non-lysogenicHaemophilus cells can be infected by DNA of lysogenic cells, thereby giving rise to phage progeny. Thus, the phage genetic material can be introduced into competentHaemophilus cells in three different ways: injection from intact phage, and infection with either phage DNA or with bacterial DNA carrying the prophage.The UV inactivation curves for infectious phage DNA and for complete phages are similar, both indicating the occurrance of host-cell reactivation. Photoreactivationin vitro of infectious phage DNA takes place to about the same high extent as observed with bacterial transforming DNA.The usefulness of this system for investigating bacterial transformation and biological effects ofin vitro treatment of DNA is discussed.with the technical assistance ofSandra J. Antoine With 4 Figures in the TextPreliminary report presented at the 7th Annual Bacterial Transformation Meeting, Aspen, Colorado, June 17–19, 1963.Supported by a travel grant from the Deutsche Forschungsgemeinschaft.Supported by Research Carreer Development Award GM-K3-7500 and Research Grant RH 00221 from the U.S. Public Health Service.