Photoreactivation and dark repair of ultraviolet light-induced pyrimidine dimers in chloroplast DNA.

A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas reinhardi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m2 of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m2 of UF light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UV- sensitive mutant, UVS1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control.     

Genetic and biochemical analysis of photolyase mutants ofChlamydomonas reinhardtii

A mutant ofChlamydomonas reinhardtii phr-1-1 is deficient in the photorepair of pyrimidine dimers in nuclear DNA but not in chloroplast DNA. In this report, a second photoreactivation-deficient strain, phr-1-2, which has a similar phenotype as phr-1-1, is described. To determine if these mutations were in different genes, complementation tests as well as tetrad analysis were performed. Six diploid strains were constructed. The diploid strains containing one mutation exhibited similar UV-light survival curves under photoreactivating conditions as the wild-type diploid, indicating recessive nature of the mutations. No increase in survival was obtained with phr-1-1 phr-1-2 compared with either the phr-1-1 phr-1-1 or phr-1-2 phr-1-2 diploids indicating a lack of complementation. The amount of DNA photolyase activity in cell-free extracts of diploids with one mutation was not significantly different from extracts of wild-type diploids indicating a lack of gene dosage. The amount of DNA photolyase activity in extracts from the phr-1-1 phr-1-2 was no greater than found in the phr-1-1 phr-1-1 or phr-1-2 phr-1-2 diploids, confirming a lack of complementation of the mutations. Analysis of 106 tetrads from a cross ofphr-1-1 arg2×phr-1-2 arg7 indicated thatphr-1-1 andphr-1-2 were mutations in the same gene.     

UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.     

Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal DNA

The induction and photorepair of pyrimidine dimers in DNA have been measured in the ultraviolet-irradiated, corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280-400 nm) induced 7.2 +/- 1.0 X 10(-5) pyrimidine dimers per kilobase (kb) of DNA per J/m2. Following 100 J/m2, 50% and greater than 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320-400 nm), respectively. In addition, approximately 70% and approximately 60% of the dimers induced by 300 and 500 J/m2, respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model with which to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.     

The mei-9a Mutant of DROSOPHILA MELANOGASTER Increases Mutagen Sensitivity and Decreases Excision Repair

The mei-9^a mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10–20 times faster than that from mei-9^a cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9^a cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.     

UV Radiation–Sensitive Norin 1 Rice Contains Defective Cyclobutane Pyrimidine Dimer Photolyase

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase–dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.     

Assay of DNA Photolyase Activity in Spinach Leaves in Relation to Cell Compartmentation-Evidence for Lack of DNA Photolyase in Chloroplasts

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.     

Assay of DNA photolyase activity in spinach leaves in relation to cell compartmentation-evidence for lack of DNA photolyase in chloroplasts

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.     

Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB-irradiated Arabidopsis leaves

Replication of Arabidopsis nuclear, mitochondrial and chloroplast DNA (ncDNA, mtDNA, cpDNA) was assayed by measuring respective changes in copies per leaf, employing quantitative PCR (QPCR) analysis with genome-specific primer pairs. All three genomes showed parallel increases during growth of cotyledons and 5th leaves in planta, maintaining approximately 13 mtDNA copies and 280 cpDNA copies per haploid nuclear genome. Detached 5th leaves, which showed good growth and DNA replication on agar plates, were irradiated at (DNA-effective) UV-B fluences of 1.3-5.0 kJ m-2 and incubated under blue (photorepair-active) plus gold light or gold light only. Under blue light, replication of all genomes after all UV fluences was approximately as efficient as replication in unirradiated leaves. UV-irradiated leaves showed little growth under gold light only; 5 kJ m-2 stopped replication of all three genomes, 2.5 kJ m-2 stopped only cpDNA replication, and 1.3 kJ m-2 only delayed cpDNA replication. Immunoassays showed that 5 kJ m-2 induced about 1.2 cyclobutane pyrimidine dimers and 0.1 [6-4]photoproducts per kbp of bulk DNA, and that both photoproducts were completely removed during 2-3 days under blue light, suggesting efficient photorepair of at least ncDNA and cpDNA. The evidence for efficient photorepair of organellar DNA contrasts with previous studies of irradiated 5-day-old seedlings, and with the apparent absence of Arabidopsis photolyases bearing transit peptides.     

Spontaneously occurring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice

Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering.     

Cloning and characterization of a class II DNA photolyase from Chlamydomonas

Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases. Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast. Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase. The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency. The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene. Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence. This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase.     

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Ultraviolet light-induction and photoreactivation of thymine dimers in a cyanobacterium,Anacystis nidulans

Partially photoreactivable mutant of Anacystis nidulans demonstrates partial photorepair of thymine dimers. The wild type which is completely photoreactivable at the conditions studied shows higher level of thymine dimer photolysis.Abbreviations UV ultraviolet light, peak intensity at 254 nm - PR photoreactivation - Dm D medium of Kratz and Myers modified by van Baalen - WT wild type     

Analysis of the plastid DNA in an Oenothera plastome mutant deficient in ribulose bisphosphate carboxylase

Summary Plastid DNA of the light green Oenothera plastome mutant sigma, from plastome I, which is deficient in ribulose bisphosphate carboxylase, has been compared with wild-type chloroplast DNA from plastome I and the related plastome IV. For this, double digestions with the restriction endonucleases Sal I, Pst I and Kpn I were used. Chloroplast DNA from plastomes I and IV differs in the sizes of several fragments, with the changes being from under 0.1 to about 0.6 Md in size. In the cleavage patterns of the mutant DNA compared to the wild-type DNA from plastome I, the only differences observed are two possible deletions of less than 0.1 Md from a fragment known to partly cover the genes for the ribosomal RNAs and from a fragment located in the small single-copy region of the molecule. It is concluded that the ribulose bisphosphate carboxylase deficiency in this mutant is not caused by a major deletion in the plastid DNA.     

Increased efficiency of photoreversal of UV-induced dimers in the DNA of chick embryo fibroblasts with post-UV dark time

The kinetics of photoreversal of UV-induced dimers in the DNA of early passage chick embryo fibroblasts was studied by monitoring disappearance of UV-endonuleae-sensitive sites. Photorepair was found to increase in efficiency when cells were incubated in the dark for several hours at 37°C following the dimer-inducing short-wavelength (254 nm) UV treatment, but prior to the photoreactivating black light (365 nm). Folllowing a UV dose of 10 J/m² it took at least 4 h in the dark to saturate this effect. This UV dose inserts roughly 2.4 dimer/10⁷ daltons of DNA. Dark repair removes about 0.08 dimers /h/10⁷ daltons. After 6 h in the dark, exposure to black light removes an additional 1.4 dimers /10⁷ daltons leaving about 0.5 dimers unaffected by this treatment. After saturation of the dark effect, the amount of photoreactivation depends only on total black light fluence and not on fluence rate for the range of rates studied. This indicates that during 30 min, the maximum time of black light exposure, no appreciable reattachment of the photorepair molecule to additional unrepaired dimer sites occurs. We estimate that the number of effective photorepair molecules per chick chick cell is at least of the order of 2 × 10⁵.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.