Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.

Summary The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3 end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 by segment from 399 to 441 by of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.     

Isolation and Characterization of Ultraviolet Light-Sensitive Mutants of the Blue-Green Alga Anacystis nidulans

Three independently isolated ultraviolet light-sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 was most sensitive to UV in the absence of photoreactivation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, indicating the presence of excision enzymes involved in dark repair. Under "black" and "white" illumination, strain uvs-1 displays photoreactivation properties nearly comparable to wild-type culture. Mutants uvs-35 and uvs-88 appeared to have partial photorecovery capacities. Upon pretreatment with chloramphenicol, photoreactivation properties of strains uvs-1 and uvs-88 were not evident although the partial photoreactivation characteristics of strain uvs-35 remained the same. Data indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.     

Radiation Genetics in Microorganisms and Evolutionary Considerations

Recent knowledge of UV-resistance mechanisms in microorganisms is reviewed in perspective, with emphasis on E. coli. Dark-repair genes are classified into "excision" and "tolerance" (ability to produce a normal copy of DNA from damaged DNA). The phenotype of DNA repair is rather common among the microorganisms compared, and yet their molecular mechanisms are not universal. In contrast, DNA photoreactivation is the simplest and the most general among these three repair systems. It is proposed that DNA repair mechanisms evolved in the order: photoreactivation, excision repair, and tolerance repair. The UV protective capacity and light-inducible RNA photoreactivation possessed by some plant viruses are interpreted to be the result of solar UV selection during a rather recent era of evolution.     

Estimation of the contribution of ionization and excitation to the lethal effect of ionizing radiation

Summary A simple theoretical model is proposed for estimating the differential contribution of ionization and excitation to the lethal effect of ionizing radiation. Numerical results were obtained on the basis of published experimental data on the ability of bacterial cellsEscherichia coli to undergo photoreactivation of radiation-induced damage. It was shown that inactivation by excitation may be highly significant for UV-hypersensitive cells capable of photoreactivation; inactivation by excitation increased with the energy of ionizing radiation and the volume of irradiated suspensions. The data are in qualitative agreement with the assumption of a possible contribution of the UV-component of erenkov radiation to the formation of excitations responsible for the lethal effect and the phenomenon of photoreactivation after ionizing radiation. Some predictions from the model are discussed.     

Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.     

The effect of post-UV dark incubation on survival of chick-embryo fibroblasts after photoreactivation

A survival assay with chick-embryo fibroblasts was used to study photoreactivation of ultraviolet (UV) irradiation-induced damage. The kinetics of the photoreactivation was studied as a function of the length of a post UV dark incubation period of from 0 to 18 h at 38.5 degrees C. The logarithmic survival curve with no photoreactivation had a Do of about 4.3 J/m2 giving approximately 0.8% relative plating efficiency after a UV dose of 21 J/m2. At this dose the efficiency of photoreactivation (survival increase per unit blacklight dose) increased with post UV incubation time reaching a maximum at 4-6 h, then declining until there was little photoreactivation observed for times longer than about 11 h. The possibility that this effect was produced by pre-UV perturbations of the cell cycle was eliminated by the fact that the same results were achieved after several rather different trypsinization protocols. The shape of the photoreactivation vs. blacklight curve at the time of peak efficiency showed a threshold up to about 3 kJ/m2, a rising portion and a plateau after 12-16 kJ/m2 when the survival increased by a factor of roughly 8.     

Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s.

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.     

Localisation of the Site of Action of tube growth stimulation by micro UV-irradiation of pine pollen

Summary Micro-irradiation of pine pollen grains was carried out with different doses at four different dose rates and the tube growth was observed. The irradiation of the whole pollen grains in the dehydrated state and dorsal position and of those in the hydrated state and ventral position induced stimulated tube growth after receiving low doses of UV light. The effect of stimulation depended on the ratio between dose and dose rate. After partial irradiation of pollen grains at low doses, carried out with the technique of blind shot, a stimulation effect could also be observed. It was calculated that the irradiation of the cytoplasm had a strong, the irradiation of the active nucleus (vegetative) had a moderate and the irradiation of the dormant nucleus (generative) had little or no dose rate dependance. The dose effect of the nuclei showed a reverse tendency to the dose effect of the cytoplasm. Experiments with different light filters suggested that the dose rate dependance of the cytoplasm is probably not caused by a repair mechanism. The vegetative nucleus showed an effect of photoreactivation and probably also of a dark repair. The generative nucleus exhibited only an effect of photoreactivation.Dedicated to Prof. Dr. Ing. H. Glubrecht on the occasion of his 60. birthday     

Evidence for Two Mechanisms of Photoreactivation in Escherichia coli B

Escherichia coli B phr^-, which is not photoreactivable under certain conditions, has been shown to exhibit photoreactivation of killing in the logarithmic growth phase at 3341 A. Dependence of the reaction upon (a) wavelength, (b) dose, and (c) dose rate of the reactivating radiation, as well as upon (d) temperature during reactivation treatment, is very similar to that of photoprotection. We conclude that this photoreactivation is similar in mechanism to photoprotection, believed to be an indirect repair process, the initial step of which is non-enzymatic and leads to a growth-division delay. We therefore call the present phenomenon “indirect photoreactivation.” Similar studies suggest that indirect photoreactivation of killing occurs also in the parent strain, E. coli B (Harm). It has often been supposed that all photoreactivation results from a photoenzymatic reaction similar to that found to operate in vitro on transforming DNA. Our data provide the first evidence for two distinct types of photoreactivation of cell killing, one of which appears not to involve photoenzymes. These experiments also show that photoprotection results from intracellular events that can be induced by treatment after, as well as before, far ultraviolet irradiation.     

Effects of caffeine and ARG7 locus on mutability of UV-treated photoreactivation-deficient mutants ofChlamydomonas reinhardtii

Forward streptomycin-resistant mutations and reverse mutations at the ARG7 locus after UV irradiation were studied in two photoreactivation-deficient mutants ofChlamydomonas reinhardtii, Phrl and Phr2. The mutant Phrl was more mutable than Phr2. Caffeine increased survival and reduced mutation rate of streptomycin-resistant mutations induced in both photoreactivation-deficient strains. Two different alleles of ARG7 locus (arg2 and arg7) were introduced into photoreactivation-deficient mutants. It was found that in the presence of both alleles, the frequency of mutants resistant to streptomycin was reduced. The reduction was more remarkable in the presence of arg2. But also under these conditions Phr1 was more mutable than Phr2.     

Loss of photoreversibility for UV mutation in E. coli using 405 nm or near-UV challenge

E. coli mutagenized with germicidal ultraviolet light (UV) were incubated to allow for development of mutation-fixation processes. Fixation was estimated from the effects on mutation frequency of photoreactivation challenge during the first 60 min post-UV. Two different light sources were used for photoreactivation, one providing effective light primarily at 405 nm and another providing a broad range of near-UV around 365 nm. Kinetics for the loss of photoreversibility (LOP) were determined. The times for completion of LOP in wild-type cells indicated one fixation process for back mutation and another for de novo or converted suppressor mutation regardless of the light source. Using 405-nm light for photoreactivation, the LOP kinetics for back mutation and de novo suppressor mutation in uvrA cells were similar. Hence, classical observations were confirmed here. Immediately post-UV all mutation frequencies were more sensitive to near-UV than 405-nm light. Experiments with rel cells supported the idea that growth delay and inhibition of induced lexA-coordinated responses may be responsible for this early, pronounced sensitivity to photoreactivation by near UV. For back mutation and de novo suppressor mutation, the sensitivity to 405-nm light was initially small and actually increased for 10-15 min. Possibly genome conformation changes are induced by UV and this affects the efficiency of photoenzymatic monomerization of 405-nm light during the first 10-15 min after irradiation.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts

Summary Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function.Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutationsThe first paper in this series is Hays et al., (1985)     

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.     

Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.     

Increase in the fraction of non-repaired mutations in Escherichia coli cells, incubated in the absence of glucose after UV-irradiation

In E. coli WP2 trpE65 cells irradiated with UV-dose of 11 J/m2, the additional small portion of induced Trp+ mutations became resistant to photoreactivation or "dark" (excision) repair after a short-termed (10-30 min) postirradiation incubation of bacteria in a minimal medium deprived of glucose and tryptophan. Since protein synthesis could not proceed in those cells because of the lack of energy and tryptophan, the data indicate that an unknown mechanism exists which imparts some mutations with the resistance to antimutagenic repair in the absence of the inducible mutagenic system. In the light of this result, one could suggest that the normal process of mutation fixation (that is the loss of sensitivity of mutations to photoreactivation or to excision repair in cells incubated in growth medium after irradiation) should not necessarily be a direct consequence of manifestation of the activity of an inducible mutagenic system.     

Photoreactivation inChlamydomonas reinhardi Dangeard

The existence of photoreactivation in the green unicellular algaChlamydomonas reinhardi Dangeard was demonstrated. The dose reduction factor was constant throughout practically the whole of the dose range and was approximately 0.5. Photoreactivation was not found in cells irradiated with X-rays. The maximum photoreactivation after ultraviolet irradiation was reached after about 30 minutes’ illumination with 2,300Lx. No difference was found between the rate of photoreactivation carried out immediately and 30 minutes after ultraviolet irradiation. The rate of photoreactivation, under the given conditions, seems to be limited chiefly by the supply of light energy. The photoreactivation enzyme is probably a stable component of the cell.     

Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis.

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.     

Human Photoreactivating Enzyme: Action Spectrum and Safelight Conditions

The action spectrum for photoreactivation by enzymes from human leukocytes and fibroblasts extends from 300 to approximately 600 nm with a maximum near 400 nm. The ability of the human enzymes to utilize light of wavelengths greater than 500 nm suggested that yellow or gold lights conventionally used as safelights for photoreactivation might serve as sources of photoreactivating light for these enzymes. Experiments using lights with a range of spectral outputs confirm that the standard yellow “safe” lights do produce photoreactivation by the human but not the Escherichia coli enzyme.     

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.     

光复活对紫外线照射大肠杆菌后突变率的影响

通过改变UV照射时间、照射后的操作速度、光复活时的温度、时间和光强度,以光复活和暗处理后细胞存活数的比值为依据,研究了不同条件下E．coli受UV照射后的光复活效应。并以E．coli对5μg/ml链霉素抗性突变率为指标,比较了不同剂量UV照射后光复活和暗处理对E．coli突变率的影响。结果表明：光复活效应在温度10℃时最明显,且与照射时间、照射后的操作速度、光复活时间和光强度成正相关;在中、低剂量UV照射后,暗处理较光复活后E．coli对链霉素抗性突变率明显高,而在高剂量下,光复活则显著高于暗处理后的突变率。