Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

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.     

UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis.

Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV-C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. V. Biochemical characterization of a strain (ebony) that is UV- and X-ray sensitive and deficient in photorepair

We investigated larval sensitivity to UV and repair of UV- and X-ray-induced lesions in the DNA of the ebony strain compared to a wild-type strain (Canton S). The ebony strain was previously characterized as being more sensitive to UV-induced killing of embryos than Canton S. Also the ebony strain is more sensitive to X-rays for induction of larval killing, dominant lethals and recessive lethals. In this paper it is demonstrated that (1) ebony larvae are more sensitive to killing by UV and less proficient in photoreactivation (PR) ability than Canton S larvae; (2) the ebony strain has a defect in PR repair of endonuclease-sensitive sites induced in the DNA of primary cell cultures by UV irradiation; (3) the ebony strain has a defect in the repair of single-strand breaks induced in the DNA by X-rays (again in primary cell cultures), at least early on in the repair incubation. A rough localization of the UV sensitivity and the PR ability is presented and the possible relevance of the biochemical to the genetic results is discussed.     

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.     

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.     

The control of dark repair mechanisms in meiotic cells

Summary The responses to UV irradiation of pre-meiotic and meiotic cells of the unicellular green alga Chlamydomonas reinhardi have been examined in a wild type and a UV-sensitive mutant (UVS1) strain. During late interphase and early prophase the cells become highly sensitive to UV under dark conditions, but show much less change in sensitivity following photoreactivation (PR). At these times the effect of PR is not dose-modifying, but very large PR factors are obtained — at the 50% survival level in some stages values up to 23 being obtained. The enhancement of dark sensitivity is attributed to a diminution in dark repair activity, and it is suggested that this is associated with the process of genetic recombination that occurs in these meiotic cells. In addition at those times when the dark repair capacity is at a low level, a phenomenon akin to UV reactivation occurs, in that within a certain dose range, survival increases with increasing dose. The possible basis of this phenomenon is discussed.     

Liquid holding recovery and photoreactivation of UV-induced damage inStreptococcus lactis

Summary Repair of ultraviolet-light-induced DNA damage inStreptococcus lactis has been examined. The wild-type strain and its derivative Lac^– possess a dark repair system (maximal increase in survival of 4-fold). Enzymatic photoreactivation exists in the two strains but a weaker photoreactivability was found in the Lac^– derivative (4 and 2-fold, respectively). Concomitant reduction of UV-induced mutagenesis (Rif^r marker) was also studied during these two repair phenomena. The absence of dark repair after saturation of photoreactivation suggests that photoreactivation is much more efficient with pyrimidine dimers as substrate.     

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.     

UV hyper‐resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase

Exposure to solar radiation can cause mortality in natural communities of pico‐phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV‐hyper‐resistant variant of high light‐adapted strain MED4. The hyper‐resistant strain was constitutively upregulated for expression of the mutT‐phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8‐oxo‐dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error‐free, in contrast to error‐prone SOS dark repair systems that are widespread in bacteria. The UV‐hyper‐resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT‐phrB operon. Two subsequent enrichments for MED4 UV‐hyper‐resistant strains from MED4 wild‐type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper‐resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

Nucleosome positioning,nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.     

UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Repair,replication and survival in UV-irradiatedEscherichia coli

The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficientEscherichia coli K-12 after a flux of 20 J m⁻². In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage ia of primary importance for excision-proficient cells which can easily cope with persisting dimers.     

Indicators for photoreactivation and dark repair studies following ultraviolet disinfection

Repair of DNA in bacteria following ultraviolet (UV) disinfection can cause reactivation of inactivated bacteria and negatively impact the efficiency of the UV disinfection process. In this study, various strains of E. coli (wild-type, UV-resistant and antibiotic-resistant strains) were investigated for their ability to perform dark repair and photoreactivation, and compared based on final repair levels after 4 h of incubation, as well as repair rates. Analysis of the results revealed that the repair abilities of different E. coli strains can differ quite significantly. In photoreactivation, the log repair ranged from 10 to 85%, with slightly lower log repair percentages when medium-pressure (MP) UV disinfection was employed. In dark repair, log repair ranged from 13 to 28% following low-pressure (LP) UV disinfection. E. coli strains ATCC 15597 and ATCC 11229 were found to repair the fastest and to the highest levels for photoreactivation and dark repair, respectively. These strains were also confirmed to repair to higher levels when compared to a pathogenic E. coli O157:H7 strain. Hence, these strains could possibly serve as conservative indicators for future repair studies following UV disinfection. In addition, dimer repair by photoreactivation and dark repair was also confirmed on a molecular level using the endonuclease sensitive site (ESS) assay.     

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.     

Repair of DNA Damage Induced by Solar UV

Solar UV radiation induces significant levels of DNA damage in living things. This damage, if left unrepaired, is lethal in humans. Recent work has demonstrated that plants possess several repair pathways for UV-induced DNA damage, including pathways for the photoreactivation of both 6-4 products and cyclobutane pyrimidine dimers (CPDs), the two lesions most frequently induced by UV. Plants also possess the more general nucleotide excision repair (NER) pathway as well as bypass polymerases that enable the plant to replicate its DNA in the absence of DNA repair.This revised version was published online in October 2005 with corrections to the Cover Date.     

Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation.

A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage.     

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage.     

Photoreactivation of UV-irradiated blue-green algae and algal virus LPP-1

Ultraviolet (UV) sensitivity and photoreactivation of blue-green algae Cylindrospermum sp., Plectonema boryanum, spores of Fischerella muscicola and algal virus (cyanophage) LPP-1 were studied. The survival value after UV irradiation of filaments of Cylindrospermum sp. and Virus LPP-1 showed exponential trend and these were comparatively sensitive towards UV than F. muscicola and P. boryanum. Photoreactivation of UV-induced damage occurred in black, blue, green, yellow, red and white light in Cylindrospermum sp., however only black, blue and white light were capable of photorepair of UV-induced damage in P. boryanum, spores of F. muscicola and virus LPP-1 in infected host alga. Pre-exposure to yellow and black light did not show photoprotection. The non-heterocystous and nitrogen fixation-less mutants of Cylindrospermum sp. were not induced by UV and their spontaneous mutation frequency was not affected after photoreactivation. The short trichome mutants of P.boryanum were more resistant towards UV.The occurrence of photoreactivation of UV-induced killing in wide range of light in Cylindrospermum sp. is the first report in organisms.