Photoreactivation Research Articles

Studies on mutagen-sensitive strains of Drosophila melanogaster I. The enhanced X-ray sensitivity of a mutant strain ( ebony) which is UV-sensitive and also deficient in photorepair

Yegorova and colleagues (1978) showed that a mutant strain of Drosophila melanogaster (ebony) was more sensitive to UV-induced killing of embryos and also less proficient in photoreactivating (PR) ability than a wild-type ( Canton-S) strain and that the genes governing UV sensitivity and PR ability were different and presumably located on the autosomes. The experiments reported in the present paper were designed to compare the patterns of sensitivity of these 2 strains and their hybrids to X-irradiation. The sensitivity of the larvae to the killing effects of X-irradiation, and of male and female germ-cell stages to the X-ray induction of genetic damage was studied. It was found that the larvae of the ebony strain are more sensitive to X-ray-induced killing than those of the Canton-S strain. The frequencies of radiation-induced dominant lethals and sex-linked recessive lethals are higher in spermatozoa sampled from ebony males than in those of Canton-S males. In spermatozoa sampled from hybrid males, the yields of dominant lethals are no higher than in those sampled from Canton-S males and do not seem to depend on the origin of the X-chromosome. There are no statistically significant differences between the ebony and Canton-S strains in the sensitivity of their spermatozoa to the induction of autosomal translocations. Stage-7 oocytes sampled from ebony females are more sensitive to the X-ray induction of dominant lethality than are those from Canton-S females; oocytes sampled from hybrid females manifest a level of sensitivity that is significantly lower than that in either parental strain. The frequencies of X-chromosome losses induced in in this germ-cell stage are significantly lower in ebony than in Canton-S females at least at the exposure level of 3000 R at which 3 experiments were carried out. There are no measurable differences in the amount of dominant lethality induced in stage-14 oocytes of ebony, Canton-S and hybrid females. When X-irradiated Berlin-K males are mated to ebony or Canton-S females, the yields of dominant lethals are higher when ebony females are used, showing that there is a “maternal effect” for this kind of damage. Such a maternal effect is also found for sex-linked recessive lethals (irradiated Muller-5 males mated to ebony or Canton-S females). However, when irradiated ring-X-chromosome-carrying males are mated to ebony or Canton-S females, the frequencies of paternal sex-chromosome losses (scored as XO males) are lower when ebony females are used. These results have been interpreted on the assumption that the ebony strain is homozygous for recessive, autosomal genes that confer increased radiosensitivity and that the Canton-S strain carries the normal, wild-type alleles for these genes. The higher yields of dominant and recessive lethals in mature spermatozoa and of dominant lethals in stage-7 oocytes are a consequence of an enhanced sensitivity to the mutagenic (in particular, to the chromosome-breaking) effects of X-irradiation and/or of defective repair of radiation-induced genetic damage. The lower yield of XO males from irradiated stage-7 oocytes of ebony females is probably a consequence of a defect in the repair of chromosome-breakage effects, resulting in the conversion of potential X losses in females into dominant lethals. The “maternal effects” for dominant lethals, sex-linked recessive lethals and for the loss of ring-X chromosomes are assumed to have a common causal basis, namely, a defective repair of chromosome-breakage events in the females of the ebony strain.

Ultraviolet irradiation of murine cytomegalovirus.

Ultraviolet irradiation of murine cytomegalovirus (MCMV) caused a rapid dose-related decline in virus infectivity, manifested by virus antigen induction, and in virus production as measured by plaque formation and infectious centre assay. The virus survival curve was multi-component, suggesting host cell-assisted reactivation. Multiplicity reactivation and photoreactivation of MCMV were not observed in these experiments. Productive infection was more sensitive to u.v. irradiation than was virus antigen production, indicating differential inactivation of virus functions. The effects of u.v. irradiation were similar in most respects to those reported for human cytomegalovirus.

Photoreactivation associated with chromosomal abnormality in sea urchin eggs fertilized with ultraviolet-irradiated sperm.

Abstract Chromosomal abnormality due to the ultraviolet (UV) irradiation of sperm and its modification by photoreactivation (PR) were studied in the sea urchin, Hemicentrotus pulcherrimus. When sperm was UV‐irradiated and allowed to fertilize unirradiated eggs, the effect of UV was manifested as an abnormal separation of chromosomes at the anaphase of the first mitosis. The frequency of abnormal anaphase increased with UV fluence. In the UV fluence of larger than 20J/m2, more than 90% of eggs showed chromosomal abnormality. The UV‐induced chromosomal abnormality was prevented by photoreactivation when the fertilized eggs were illuminated with visible light. The PR sector for the PR of chromosomal abnormality, which was comparable to the PR sector for morphological abnormality, was higher than that of cleavage delay. The UV sensitivity expressed in terms of chromosomal abnormality was significantly higher than the sensitivity expressed in terms of morphological abnormality. Photo‐reactivation illumination sufficed to effect a nearly complete PR when applied up to 20 min after fertilization, while the PR effectiveness declined thereafter. Illumination after 40 min had little effect for PR.

The role of pyrimidine dimers in postreplication repair in Neurospora.

Using the Micrococcus luteus dimer specific endonuclease assay of Wilkins (1973), and photoreactivation we have examined the induction and fate of ultraviolet induced pyrimidine dimers in the excision defective strain, uvs-2, of Neurospora crassa. Dimer induction was fluence dependent from 0 to 800 ergs/mm2 UV. An interdimer distance of 19.6 x 10(6) DNA molecular weight was found after a fluence of 220 ergs/mm2. We confirm the earlier report that this mutant is completely excision defective (Worthy and Epler 1972). Photoreactivation (PR), which greatly enhanced survival (by 10 fold after 440 ergs/mm2 UV), reduced significantly (40-44%) the number of UV-endonuclease sensitive sites found in irradiated DNA. This treatment also alleviated immediately some of the temporary blocks to high molecular weight DNA synthesis (elongation or ligation) seen in irradiated cells. We have also attempted to elucidate the mechanism of cellular postreplication repair used to overcome the UV inhibition to DNA synthesis. It was determined that during postreplication repair, Neurospora does not use recombination to bypass dimers and that single stranded DNA gaps opposite dimers do not appear to be present during the time when DNA being synthesized is made only in short pieces.

Loss of photoreactivation in UV-irradiated cultured fish cells under different conditions.

Abstract— CAF‐MM1 cells derived from a goldfish have photoreactivability for the damage induced by ultraviolet light (UV). When UV‐irradiated cells were incubated in the dark at 26AoC, the longest interval in which photoreactivation (PR) was observed (i.e. effective time for PR), measured by colony formation technique, was about 30 h after the UV irradiation. However, if the cells were incubated at 20AoC, the effective time was prolonged. Since each time appeared to correspond to the doubling time of the cells at each temperature, the loss of photoreactivability is suggested to be closely related to cell growth or progression of cell cycle. The loss of PR was not observed in the cells held in confluence up to 48 h after UV irradiation, in support of the above suggestion. Photoreactivating enzyme in growing CAF‐MM1 cells incubated in the dark for 24 h after UV irradiation was shown to be active, so that it is not possible that the cause of the loss of PR is change in the activity of photoreactivating enzyme.

Light-flash analysis of the photoenzymic repair process in yeast cells III. The photoenzymic repair of respiratory-sufficient and respiratory-deficient cells cultured aerobically and anaerobically

In the previous papers, determinations of the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per cell and of the rate constant for the formation of the PRE-pyrimidine dimer complex, k 1, for yeast cells cultured aerobically, were described. In the present work, the number of PRE molecules and ther ate constant k 1 for respiratory-sufficient (RS) and respiratory-deficient (RD) cells of yeast cultured anaerobically were determined. The numbers of PRE molecules in both RS and RD cells cultured anaerobically were larger than those in cells cultured aerobicaly, whereas the rate constant k 1 (nuclear volume). (PRE molecules) −1 sec −1 showed a reverse relationship. The numbers of PRE molecules in cells cultured aerobically in various media were also determined.

Photoreactivation associated with morphological abnormality in sea urchin embryos induced by ultraviolet-irradiated sperm.

Abstract— Morphological abnormality due to the UV irradiation of sperm and its modification by photoreactivation (PR) were studied in the sea urchin, Hemicentrotus pukherrimus. When sperm was UV‐irradiated and allowed to fertilize unirradiated eggs, the effect of the UV was manifested as an abnormal morphology of embryos in the gastrula or later stages. The UV‐induced morphological abnormality was prevented by photoreactivation when the fertilized eggs were illuminated with visible light. In the experiments on a stage‐dependent change of PR effectiveness, it was found that an illumination sufficed to effect a nearly complete PR when applied up to the onset of the first DNA synthetic phase, while the PR effectiveness declined thereafter. Illumination after the completion of DNA synthesis had little effect for PR.

Photoreactivation of ultraviolet-irradiation damage in Staphylococcus aureus.

This study reports the capacity of Staphylococcus aureus strain 7-8 to undergo photoenzymatic repair of UV-irradiation induced damage and compares it to the photoreactivation (PR) response of Escherichia coli strain B. Staphylococcus aureus showed greater inhibition by UV irradiation than E. coli, consistent with its higher adenine and thymine content of DNA. Staphylococcus aureus showed an enhanced rate of photoreactivation with no lag in initiation of the PR response at low PR doses compared to E. coli. Maximum PR capacity of both cultures was about equal and occurred in cultures incubated at 23-25°. The PR responses at 11-12 and 35-37° for S. aureus and E. coli differed although both were capable of PR at each of these temperatures. The PR response of E. coli was directly related to the dosage of PR light (J/m2); however, the photoenzymatic capacity of S. aureus was not directly responsive to continued decrease in light intensity. The capacity of S. aureus to undergo liquid holding recovery (LHR) occurred at 23-25° (not at 11-12° or 35-37°), whereas E. coli underwent LHR at 11-12° and 23-25° but not at 35-37°. The LHR response of S. aureus was somewhat more effective than E. coli and did not show the direct response to increased liquid-holding period as did E. coli.

Light-flash analysis of the photoenzymic repair process in yeast cells. II. Determination of the rate constant for formation of photoreactivating enzymes-pyrimidine dimer complexes and its activation energy term.

As reported in the previous paper, the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per yeast cell was determined by the use of intense light flashes. In the present work, the reaction rate constant for the formation of PRE-substrate complexes, k1, and the activation energy term of k1 were determined by yeast cells in vivo by the use of light flashes. At 30 degrees C, k1 equalled (6.5 +/- 1.1) x 10(-5) (nuclear volume) x (molecule)(-1) sec(-1), which corresponded to 1.1 x 10(5) 1 mole(-1) sec(-1), on the assumption that a nuclear volume is 3 x 10(-15) 1. k1 showed positive temperature dependence as described by the arrhenius expression with an activation energy of 11.8 +/- 1.6 kcal mole(-1).

Mechanisms of DNA Replication and Mutagenesis in Ultraviolet-Irradiated Bacteria and Mammalian Cells

This chapter discusses the studies on DNA replication in bacteria and mammalian cells irradiated by ultraviolet (UV) light. This topic is of interest because cellular mechanisms for replicating damaged DNA are functionally related to cellular control of UV-light-induced mutagenesis. The chapter restricts its analysis to UV damage, since a lesion in DNA that results in major biological and mutagenic effects, the pyrimidine dimer, has been identified. Other pathways for the repair of pyrimidine dimers (e.g., excision repair and photo reactivation) have been extensively reviewed and are mentioned briefly in the chapter. In particular, the chapter also discusses the effects of UV irradiation on DNA synthesis in bacteria, mammalian cells, bacteriophages, and animal viruses. Possible mechanisms for replication of damaged chromosomes are described, and the mechanisms of UV-induced mutagenesis are analyzed. Because studies performed in bacteria have frequently served as models for similar processes in mammalian cells, the bacterial studies are reviewed first. The extent to which these models apply to mammalian cell systems is discussed, and where appropriate new models are presented to explain data from mammalian cells that fail to be explained satisfactorily by the bacterial models.

Hertwig effect caused by UV-irradiation of sperm of Oryzias latipes (teleost) and its photoreactivation

When sperm of the fish Oryzias latipes were irradiated with ultrviolet light and allowed to fertilize normal egges, the so-called “Hertwig effect” was observed, with a dose-dependent decrease in survival rate at low doses (0–27 J · m −2) but a better survival rate at higher dose ranges. Illumination with visible light after fertilization (10–70 min after insemination) showed the existence of photoreactivation (PR), demonstrating that pyrimidine dimers are a lesion in sperm DNA that is mainly responsible for the UV-caused Hertwig effect. Genetic analysis, in which sperm from a wild-type of this fish was used, showed that, after UV-irradiation at the high dose range, male nuclei did not participate in embryonic development (a gynogenetic haploid condition). Embryos having only a maternal set of chromosomes could develop no further than stage 27. Only the visible light during the early part (until around 20–30 min after insemination, at 25°C) of the single-cell stage was effective for PR; illumination thereafter was not.

Damage and repair in mammalian cells after exposure to non-ionizing radiations II. Photoreactivation and killing of rat kangaroo cells ( Potorous tridactylus) and herpes simplex virus-1 by exposure to fluorescent “white” light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridactylus) has been studied at wavelengths > 375 nm from either fluorescent “white” light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths > 475 nm, and even > 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The “fluence reduction factor” of PR, which is >3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight >375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.

Regeneration and photoreactivation after ultraviolet irradiation of moss cells

Three species of moss, Funaria hygrometrica Hedw., Physcomitrium pyriforme (Hedw.) Hampe, and Physcomitrella patens (Hedw.) BSG (Funariaceae) were examined for (a) the presence of a photoreactivating (PR) repair mechanism after ultraviolet (u.v.) irradiation and (b) the time necessary for photoreactivation to occur. No PR of regeneration was detected in either Funaria hygrometrica or Physcomitrium pyriforme; however, Physcomitrella patens did show significantly increased survival when cultures were exposed to visible light after u.v. irradiation. The length of time necessary for repair in visible light depended on the magnitude of initial u.v. exposure.

Photoreactivation of Ionizing-radiation-induced Damage inE. Coli

Photoreactivation of Ionizing-radiation-induced Damage in<i>E. Coli</i>

Enzymatic Photoreactivation of Escherichia coli after Ionizing Irradiation: Chemical Evidence for the Production of Pyrimidine Dimers

By comparing the results for a uvrA recAand a uvrA uvrB recA phr strain, we conclude that the photoreactivation (PR) observed after ionizing irradiation involves the same enzyme that is responsible for the PR of ultraviolet (uv) radiation-induced cyclobutadipyrimidines (Py Py). A comparison of the photoreactivable sectors in a uvrA recA strain after uv irradiation and 137Cs γ irradiation indicates that 10 krad of γ radiation produces about 0.07 Jm−2-equivalents of 254-nm radiation-induced photoreactivable damage. After 400 krad, an acid hydrolysate of the isolated DNA revealed the presence of thymine-containing Py Py, as evidenced by their chromatographic properties and their photochemical reversibility. The yield of thymine dimers after 400 krad of γ irradiation, as detected chromatographically, was in close agreement with the yield that was predicted from our PR studies. Thus, the PR observed after ionizing irradiation in strains that are blocked in excision repair and in postreplication repair is d...

Cloning of the phr gene and amplification of photolyase in Escherichia coli

A new technique is developed for physically enriching recombinant DNA molecules in an in vitro recombination mixture. UV-irradiation of the donor DNA before recombination enables photoreactivating enzyme (PRE) (deoxyribodipyrimidine photolyase, EC 4.1.99.3) to attach to the donor segments in recombinant molecules. This attached protein causes retention of the recombinant molecules on a nitrocellulose filter, while molecules not containing donor DNA pass through. The bound DNA is repaired of its UV damage and released for insertion into cells by exposure to photoreactivating light in situ, yielding approx. 350-fold enrichment. Although applicable to any gene, this procedure has been used in cloning the Escherichia coli phr gene itself, permitting 100-fold amplification of the gene product in vivo.

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.

The Absence of an Effect of Photoreactivation on Sub-lethal Damage Accumulation in a Photoreactive Wallaby Cell-line

Monolayer cultures of JU56 wallaby cells were exposed to germicidal U.V. and/or photoreactivating (PR) light. The U.V. exposures induced dose-dependent cell-death. The survival data are consistent with a common extrapolation number (n) of 6 x 17 +/- 0 x 98 with a D(0) of 123 x 0 +/- 6 x 8 erg/mm2 for photo-reactivated cells and a D0 of 87 x 3 +/- 4 x 9 erg/mm2 for non-photoreactivated cells; the photoreactivation protected the cells with a dose-modification factor of 1 x 41 +/- 0 x 02. Therefore PR is not a shoulder phenomenon and so has no relationship to the repair of sub-lethal damage.

Lethal effects of pyrimidine dimers induced at 365 nm in strains of E. coli differing in repair capability.

Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 AB2480 and K12 AB2463 indicating a significant role of pyrimidine dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 AB1157, after 365-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-enzymatic repair after fluences of 365-nm radiation of 2 X 10(6) J/m-2 or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.

An unusual case of photoreactivation observed in an insect egg (Smittia Spec., chironomidae, diptera).

Abstract— An action spectrum for inactivation by ultraviolet (UV) radiation of Smittia eggs during intravitelline cleavage was established, taking into account wavelength‐dependent shielding of the effective targets. Under the assumption of a random distribution of the effective targets in the egg, the action spectrum displayed only one very distinct peak at 295 nm. The eggs were photoreactivable with an action spectrum similar but not identical to that found for direct photoreactivation (PR) in E. coli Indirect PR seems not involved because light was effective only after but not before UV. Temperature dependence and dose rate saturation could not be observed. The photoreactivable sector (PRS) was 0.75 after UV inactivation at 295 nm but only 0.32 after UV inactivation at 265 nm. Initial PR rates were highest after 295 nm and lowest after 265 nm. During migration of cleavage nuclei into the periplasm, when the shielding of nuclei by yolk material decreases by an order of magnitude, no corresponding increase in the sensitivity of the eggs to UV was observed. After inactivation at the blastoderm stage, when the nuclei are no longer shielded by yolk material, the PRS was also high (0.79) after UV of 295 nm but again lower (0.59) after 265 nm. These data are difficult to understand within the conceptual framework of light‐dependent enzymatic splitting of UV‐induced pyrimidine dimers in nucleic acids. Yet this type of PR seems to play a vital role in the survival of Smittia eggs under sunlight without need for pigmentation or shading.