Photorepair and excision repair removal of UV-induced pyrimidine dimers and (6-4) photoproducts in the tail fin of the medaka, Oryzias latipes

Induction and repair of UV-B induced DNa damage in the tail fin of the Medaka, were examined immunohistochemicaly and by the enzyme-linked immunosorbent assay (ELISA). UV-induced DNA damage was detected only in the outermost layer of epithelial cells and did not differ in fishes having different degree of melanization. Both pyrimidine dimers and (6-4) photoproducts in the fin cells were removed by excision repair in the dark, the excision of (6-4) photoproducts being about twice as efficient as that of pyrimidine dimers. The rate of excision repair of UV-induced lesions in fin tissue was three to four times that in cultured Medaka cells, OL32. In the fin cells, reductions in the numbers of pyrimidine dimers and (6-4) photoproducts were seen after treatment with fluorescent light, whereas less reductions of pyrimidine dimers and no reductions of (6-4) photoproducts were observed in OL32 cells.     

Kinetics of repair of UV-induced DNA damage in repair-proficient and -deficient cells as determined by quantitative polymerase chain reaction

Advances in methodologies to monitor gene-specific repair in human cells have facilitated a detailed understanding of the complexity of the nucleotide excision repair system. One of these procedures, quantitative polymerase chain reaction (QPCR), holds significant promise for dissecting the fine structure of the repair of UV-induced DNA damage. This assay was used to study the repair of UV photoproducts in both actively transcribed and nontranscribed genes from human cells that were capable of (1) repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts; (2) removal of neither dimers nor 6-4 photoproducts; (3) strong preferential repair of 6-4 photoproducts relative to dimers; and (4) severely depressed rates of 6-4 photoproducts and dimers. Detailed kinetic analyses revealed that repair of both active and inactive genes can be studied with a very fine degree of precision and that the repair status of the cells can easily be detected by use of the procedures described.     

UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana

Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and independent pathways. The mechanism of the "dark repair" pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined. Four of these mutants are defective in the dark repair of UV-induced pyrimidine [6-4]pyrimidinone dimers. These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product. The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi.     

Photoreactivation in bacteria and in skin

In many procaryotic and eucaryotic cells, photoreactivating enzyme mediates light-dependent repair of UV-induced damage: the enzyme binds to a pyrimidine dimer in DNA, and, on absorption of a photon (300-600 nm), specifically monomerizes the dimer, thus repairing the DNA. Photoreactivating enzyme has been found in human tissues and human cells in culture; human cells in culture can photoreactivate cellular dimers, and can mediate photoreactivation of Herpes (human fibroblasts) and Epstein-Barr virus (human leukocytes). Measurements of pyrimidine dimer formation and repair in human skin indicate that detectable numbers of dimers are formed at 1 minimal erythemal dose, that the dimers are rapidly removed in skin kept in the absence of light, and they are more rapidly removed when the skin is exposed to visible light.     

Proteins that participate in nucleotide excision repair of DNA in mammalian cells

The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.     

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.     

The fission yeast UVDR DNA repair pathway is inducible

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.