Effect of photoreactivating light on survival of ultraviolet-light-irradiatedStreptomyces lividans 66

Summary Biological systems can repair damage induced in their DNA by ultraviolet light (UV). Most cells contain at least three DNA repair pathways, each of which has a marked effect on UV survival. Excision repair and recombinational (postreplication) repair are light-independent whereas photoreactivation (PR), whether enzyzmatic or photochemical, is light-dependent. The specificity of photoreactivation for UV-induced DNA damage allows it to be used as a tool for examining whether premutational DNA lesions are preferred sites for photoreversal; it therefore plays an important role in mutagenesis studies. Evidence is presented here that PR occurs in a time-dependent fashion in three strains ofStreptomyces lividans 66. The effect appears to be independent of temperature and is observed only when PR treatment is given after UV irradiation. The present experiments do not discriminate between enzymatic and photochemical protection.     

Genetic Effects of Uv Irradiation on Excision-Proficient and -Deficient Yeast during Meiosis

The lethal and recombinational responses to ultraviolet light irradiation (UV) by excision-proficient (RAD⁺) and deficient strains (rad1) of Saccharomyces cerevisiae has been examined in cells undergoing meiosis. Cells that exhibit high levels of meiotic synchrony were irradiated either at the beginning or at various times during meiosis and allowed to proceed through meiosis. Based on survival responses, the only excision repair mechanism for UV damage available during meiosis is that controlled by the RAD1 pathway. The presence of pyrimidine dimers at the beginning of meiosis does not prevent cells from undergoing meiosis; however, the spore products exhibit much lower survival than cells from earlier stages of meiosis. The reduced survival is probably due to effects of UV on recombination. Meiotic levels of gene conversion are reduced only two to three times in these experiments; however, intergenic recombination is nearly abolished after a dose of 4 J/m ² to the rad1 strain. Exposure to 25 J/m² had little effect on the wild-type strain. Since normal meiotic reciprocal recombination is generally considered to involve gene conversion-type intermediates, it appears that unrepaired UV damage dissociates the two processes. These results complement those obtained with the mei-9 mutants of Drosophila which also demonstrate a dissociation between gene conversion and reciprocal recombination. These results are consistent with molecular observations on the UV-irradiated rad1 strain in that there is no excision of pyrimidine dimers or exchange of dimers during meiosis.     

Developmental changes in the sensitivity of Volvox to ultraviolet light

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m²) and subjected to variable lenght periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m²) is a nonlinear increase in cytotoxicity and is disproportionanately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.     

Studies of Chloroplast Development in Euglena: XIV. Sequential Interactions of Ultraviolet Light and Photoreactivating Light in Green Colony Formation

Photoreactivation (PR) of green colony-forming ability in Euglena is pH-insensitive from pH 6.0 to 8.0 and temperature-sensitive with a maximum rate at 35°C. There is no PR at 0°C. The rate of PR varies with the growth stage of the cells; PR of exponential phase cells is slower than that of stationary phase cells. The reciprocity rule holds for PR over a 6-fold range of intensity. The shape of PR curves is a function of the UV dose; there appears to be a progressive increase in multiplicity until a limiting multiplicity is reached as indicated by the fact that curves for high doses are superposable. Dark-grown and light-grown cells give the same PR response for comparable UV doses. UV inactivation of cells which have been treated with UV and then with PR light shows that, if the PR dose is sufficiently large, the same UV-inactivation curve is obtained as for nonpretreated control cells. Doses of PR lower than the saturating dose produce UV-inactivation curves, the ultimate slopes of which are parallel to the slope of the control curve, but which show reduced multiplicity. The multiplicity of these curves increases with increasing PR dose. The UV inactivation of green colony-forming ability in Euglena is completely photoreactivable at the doses studied, in contrast with the UV inactivation of colony-forming ability, which occurs at considerably higher UV doses and behaves like most other photoreactivable systems, showing a photoreactivable sector of 0.32.     

Temperature-sensitive photosynthesis-deficient mutants ofAnabœna variabilis show enhanced ultraviolet sensitivity and loss of repair mechanism

Six temperature-sensitive mutants derived from the cyanobacteriumAnabœna variabilis exhibited differences in their photosynthetic efficiency (as evidenced by oxygen evolution studies). All the ts-mutants exhibited lower chlorophyll and phycocyanin contents at 40°C relative to the wild strain and to their control cultures at 28°C. Whole cell absorption spectra of the wild strain showed the same level of chlorophyll, phycocyanin and phycoerythrin at 28 and 40°C, while the spectra from UV irradiated cells showed a decreased content of these pigments. The UV-sensitivity, photoreactivation and dark repair of the ts-mutants indicated a four- to seven-fold increased sensitivity to UV-light as evidenced by LD₃₇ values. The ability of these six mutants to repair UV-induced lesions either by photoreactivation or dark-repair was lower than in the wild strain. The ability of ts-43 and ts-49 to mediate dark-repair appears to have been lost, as documented by the survival curves obtained after post-irradiation treatment with caffeine. These results point to a relationship between the photosynthetic efficiency and the ability to repair UV-induced lesions.     

Dark recovery of UV-irradiated phage T1 I. A minor recovery effect whose exclusion permits the study of survival kinetics under presumably repairless conditions

The survival of UV-irradiated phage T1 is much lower in excision repair-deficient than in excision repair-proficient E. coli cells, due to lack of “host cell reactivation” (HCR). An additional decrease in phage survival occurs when repair-deficient (HCR⁻) host cells have been exposed to UV doses from 3000–10 000 erg mm⁻² of 254 nm UV-radiation prior to infection. The observed effect is attributed to loss of a minor phage recovery process, which requires neither the bacterial excision repair nor the bacterial REC repair system. This type of recovery is little affected by caffeine or acriflavine at concentrations that preclude HCR completely. Its full inhibition by UV-irradiation of the cells requires an approximately 8 times larger dose than complete inhibition of HCR.

In heavily preirradiated cells, the T1 burst size is extremely small and multiplicity reactivation is considerably less extensive than in unirradiated cells. Presumably the survival of singly infecting T1 in these cells reflects absence of any type of repair. The observed phage sensitivity and shape of the curve are compatible with the expectation for completely repairless conditions. The mechanism underlying the minor recovery is not known; theoretical considerations make a phage REC repair mechanism seem likely.     

Studies of Chloroplast Development in Euglena: XV. Factors Influencing the Decay of Photoreactivability of Green Colony Formation

When UV-treated cells of Euglena gracilis var. bacillaris are incubated in the dark in a nutrient medium which permits cell division, they lose the ability to be photoreactivated. The rate of this loss increases with the UV dose. For any given UV dose, the rate of decay increases with increasing growth rate. The same phenomena are observed in light-grown and in dark-grown cells, although the sensitivity to UV of the light-grown cells is smaller by a factor of 1.7. The kinetics of photoreactivation (PR) change during the decay of photoreactivability only if the cells are incubated in growth medium. A UV-inactivation curve for cells photoreactivated only after appreciable PR shows the same slope as that for untreated cells (number of UV-sensitive targets). These results are discussed from the point of view of possible models.     

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.     

Electiveness of photorepair, influence of dark-repair on shape of dose-response curves, and high-dose decline, in UV-induced colour mutations of Serratia.

Strain CV of Serratia marcescens mutates by UV with high frequency to 3 groups of mutants (w, h, s) differing in colour from the red wild-type. The mutational dose—response curve has a curvature corresponding to about 3 hits. It reaches a peak and declines at high doses. Inactivation curves have a broad shoulder and mostly, but not always, a break to a lesser slope at UV doses near the peak of mutations. Photoreactivation (PR) gives a dose reduction of about 2 for both inactivation and mutation including the break and peak. The dose curve with PR for w-mutations shows 1 hit-, and the other types 2-hit curvature leading to a change of mutation spectrum with dose due to PR. The UV-sensitive mutant uvs21 of CV has a survival curve with a small shoulder and a long upward concavity without a break, and the mutation curve is of the one-hit type without a peak and decline. PR gives a dose reduction of 12 for inactivation and of 7.5 for mutation. The 3-hit mutation curve of CV is interpreted by assuming that 2 further hits are required to protect the 1-hit pre-mutations from being abolished by the repair lacking in uvs21. UV induction of SOS repair cannot be responsible for the 3-hit curvature because UVR of phages and induction of prophage are already saturated at rather low doses. High-dose decline (HDD) of mutations in CV is probably caused neiher by a fraction of UV-resistant cells in the population nor by post-mutational selective inhibition of growth of mutants by UV-inactivated neighbour cells. As HDD is not observed in uvs21, possibly the non-mutagenic repair lacking from uvs21 interferes with the mutation finishing processes at high doses in the repair-proficient strain CV. However, UV induction of this interference cannot be a one-hit process but requires a very large number of hits.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Liquid-holding and recovery from UV-induced damage in Eudorina elegans

Summary Eudorina elegans does not respond to liquid-holding or to postirradiation medium effects by changes in recovery.A decrease in survival ability is observed if a culture is starved prior to irradiation, or is incubated at 22°C rather than 32°C following UV irradiation. Eudorina loses the ability to photoreactivate UV damage within 10 to 48 h following irradiation, depending upon the pre-and post-UV culture conditions.The results are interpreted as indicating a failure of Eudorina elegans to carry out specific dark repair of UV damage. Some reactivation may occur during cellular DNA synthesis.Abbreviations used PR photoreactivation - LHR liquid holding recovery - LHP liquid holding protection - ERR excision-resynthesis-repair - BC complete medium - BM minimal medium - cfa colony forming ability - cfu colony forming units Supported by grants from the National Research Council of Canada # A4431.     

Enhancement of repair of UV-irradiated plasmids in cultured fish cells by fluorescent light preillumination and growth arrest.

The UV-irradiated plasmid pBSCATSV, which could express chloramphenicol acetyltransferase (CAT) in the presence of SV40 early promoter, was transfected into RBCF-1 cells derived from the goldfish (Carassius auratus). The cells were incubated in the dark for 24 h and then the CAT activity was measured. CAT expression relative to non-irradiated control was calculated. The CAT expression of the exponentially growing cells transfected with UV-irradiated plasmid was enhanced by fluorescent light (FL) preillumination of the cells 8 h before transfection. The efficiency of photorepair (PR) measured by CAT expression was also enhanced by the same FL preillumination. This suggests that FL preillumination enhances both photorepair and dark repair of RBCF-1 cells for UV-damaged plasmid transfected into the cells. The enhancement of repair of UV damage by FL preillumination was also observed in survival assays. When the UV-irradiated pBSCATSV was transfected into growth-arrested cells in confluent culture, CAT expression was less sensitive to UV irradiation, and FL preillumination was much less effective in enhancing photorepair and dark repair.     

Uv-inducible repair II: its role in various defective mutants of Escherichia coli K-12.

Summary Involvement of UV-inducible protein(s) in repair of various E. coli K-12 cell strains has been investigated using a procedure of double UV irradiation and postincubation with chloramphenicol.From the course of dose survival curves the following conclusions concerning significance of a UV-inducible protein have been drawn: 1. It is very improtant for wild type cells; in these cells its early occurrence is necessary to prevent killing. 2. It is involved in repair of excision-deficient cells; however, its action early after UV is less urgent. 3. It is not involved at all in reapir of lex mutant cells; 4. It exhibits some effect on survival of recA as well as recB mutant cells.We conclude that the protein is involved in excision repair as well as in resumption of DNA replication.     

Dependence of the U.V. Survival of transforming DNA on the amount of DNA uptake per cell

Summary UV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the marker survival is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10^-3 to 2.5×10^-2 g/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system.It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5g/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.Dedicated to Professor H. Nachtscheim on the occasion of his 80th birthday.     

Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.     

Developmental changes in the sensitivity of Volvox to ultraviolet light

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m2) and subjected to variable length periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m2) is a nonlinear increase in cytotoxicity and is disproportionately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.     

The repair of UV-irradiated plasmids transfected into cultured fish cells

UV-irradiated plasmids (pNPV B and pBSCATSV) were transfected into RBCF-1 cells derived from a goldfish (Carassius auratus) and into a xeroderma pigmentosum (group A) cell line, XP20SSV. The frequency of stable neor transformation by pNPV B decreased in a dose-dependent manner. However, in spite of large differences in UV sensitivity detected in the colony formation assay, the dose-response curves of RBCF-1 cells and XP20SSV cells were almost the same. The photorecovery (PR) of transforming activity of UV-irradiated plasmids was confirmed in RBCF-1 cells but its extent was much smaller than that observed in the survival assay. The expression of the transfected cat (chloramphenicol acetyltransferase; CAT) gene after 24-h incubation in the dark was much more sensitive to UV irradiation when compared with the stable transformation assay. The extent of PR of cat gene expression in RBCF-1 cells was high and comparable with that of the survival assay. The CAT value of RBCF-1 cells transfected with UV-irradiated plasmids relative to that of unirradiated controls increased as incubation time in the dark after transfection became longer. This suggests that the UV lesions on the plasmids transfected in the RBCF-1 cells were repaired in the dark. The cat gene expression of UV-irradiated plasmids in XP20SSV was very low and independent of incubation time after transfection.     

Photoreactivation of cells and phages injuried by ultraviolet radiation in the ecological long-wave band]

Photoreactivation (PR) was measured after inactivation by far (254 nm), middle (300-315 nm) and near (315-400 nm) UV radiation of Paramecium caudatum and 8 strains of Escherichia coli differing in PR and dark repair capability. PR volume was high and practically the same after irradiation by far and middle UV, but PR was not observed in near UV-inactivated cells of all the strains. It is proposed that pyrimidine dimers are not significant in near UV lethal lesions in cells, as near UV-irradiated phages (T7 and lambdacI 857) are not photoreactivated in undamaged host bacterial cells.     

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.