Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.     

Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro

The main lesion produced in DNA by UV-C irradiation of spores of Bacillus subtilis is 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP). Using high-performance liquid chromatography-mass spectrometry analysis of the photoproducts, we showed that in wild-type B. subtilis spores (1) UV-C irradiation generates almost exclusively SP with little if any CPD and 6-4PP; (2) the SP generated is approximately 99% of the intrastrand derivative, but approximately 1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV-C irradiation of spores lacking the majority of their alpha/beta-type SASP gave less SP than with wild-type spores and significant levels of CPD and 6-4PP. The binding of an alpha/beta-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6-4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

Induction, distribution and repair of UV photodamage in the platyfish, Xiphophorus signum

The genus Xiphophorus is an important model for investigating the etiology and genetics of sunlight-induced melanoma as well as other cancers. We used immunological techniques to determine the induction, distribution and repair of cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone dimers ([6-4]PD) in different tissues of Xiphophorus signum exposed to ultraviolet-B light. We found that the (6-4)PD was induced at 5 to 10-fold lower frequency than the CPD and that scalation provided considerable photoprotection against both photoproducts. Photoenzymatic repair (PER) was very efficient in X. signum with most of the lesions removed within 20 min; PER of CPD occurred at about twice the rate of (6-4)PD. Nucleotide excision repair (NER) was much less efficient than PER and the rates of CPD and (6-4)PD removal were comparable. PER was more efficient in the caudal fin compared to the lateral epidermis; the opposite was true for NER. Although the initial rate of CPD excision was five-fold faster in the lateral epidermis compared to the caudal fin a considerable amount of residual damage remained in both tissues. The diverse photochemical and photobiological responses observed in X. signum suggest that heritable traits governing deoxyribonucleic acid damage induction and repair may be involved in the susceptibility of other Xiphophorus species to melanomagenesis.     

Pyrimidine (6-4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR

UVC irradiation of genomic DNA induces two main types of potentially mutagenic base modifications: cyclobutane pyrimidine dimers (CPDs) and the less frequent (15-30% of CPD levels) pyrimidine (6-4) pyrimidone photoproducts (6-4PP). Ligation-mediated PCR (LMPCR), a genomic sequencing technique, allows CPD mapping at nucleotide resolution following irradiation with sublethal doses of UVB or UVC for most cell types. In contrast, a dose of 80 J/m(2) of UVC that is lethal for the majority of cell types is necessary to map 6-4PP by the LMPCR technique. This compromises the use of LMPCR to study the repair of 6-4PP. To date, no other techniques have been developed to study 6-4PP repair at nucleotide resolution. We have therefore adapted a recently developed technique for the mapping of 6-4PP: terminal transferase-dependent PCR (TDPCR). TDPCR is in many ways similar to LMPCR. This technique is more sensitive and allows the mapping of 6-4PP at UVC doses as low as 10 J/m(2) in genomic DNA and in living cells.     

Evaluation of photolyase (Photosome) repair activity in human keratinocytes after a single dose of ultraviolet B irradiation using the comet assay

Photosome is constituted of photolyases included in liposomes. Photolyase is a bacterial enzyme that can repair ultraviolet B (UVB)-induced cyclobutane pyrimidine dimers (CPD) in eukaryotic cells. A modified version of the alkaline comet assay has been set up to evaluate the repair activity of this enzyme after a single dose of UVB (312 nm, 0.06 J/cm2) in human keratinocytes. The formation of single strand breaks (SSB) induced by the UVA photoactivation of the enzyme (1.2 J/cm2) was inhibited by the pretreatment of the cells with 4 mM L-ergothioneine (ERT) during 30 min at 37 degrees C. To increase the sensitivity of the comet assay, an additional lysis was used with a buffer containing sodium dodecyl sulfate (0.5%) and proteinase K (0.1 mg/ml) for 60 min at 37 degrees C. Unrepaired CPD by photolyase were revealed by a second enzymatic treatment with T4 endonuclease V, a CPD specific glycosylase. UVB irradiation increased the SSB level in keratinocytes and additional T4NV treatment enhanced this SSB level by 1.5-2.0-fold confirming that CPD were the major base modifications generated by UVB irradiation. UVA-photoactivated Photosome repaired CPD lesions and decreased the SSB levels by 2.6-3.3-fold. Photosome could be an additional component of sunscreens to reduce the development of skin cancer.     

Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.     

Photoregulation of DMA Photolyases in Broom Sorghum Seedlings

Abstract— Using dark-grown 3 day-old Sorghum bicolor seedlings, photorepair of cyclobutane pyrimidine dimers (CPD) by CPD-photoIyase and pyrimidine-(6–4)pyrimidinone photoproducts ([6–4]PP) by [6–4]PP-photolyase was studied in vivo and in vitro. The two types of DNA photoproducts were measured with specific monoclonal antibodies. Sorghum seedlings have different photorepair enzymes for CPD and [6–4]PP. The CPD-photolyase was found to be increased in response to light with maximal efficiency in the UVA/blue spectral range, whereas [6–4]PP-photolyase was not influenced by light.     

UNIQUE DNA REPAIR PROPERTY OF AN ULTRAVIOLET-SENSITIVE (radC MUTANT OF Dictyostelium discoideum

Abstract— Dictyostelium discoideum is an organism that shows higher UV resistance than other organisms, such as Escherichia coli and human cultured cells. We examined the removal of cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts from DNA in the radC mutant and the wild-type strain using an enzyme-linked immunosorbent assay with monoclonal antibodies. Wild-type cells excised more than 90% of both CPD and 6–4 photoproducts within 4 h. Dictyostelium discoideum appeared to have a special repair system, because 6–4 photoproducts were repaired faster than CPD in E. coli and human cultured cells. In radC mutant cells, although only 50% of CPD were excised from DNA within 8 h, effective removal of 6–4 photoproducts (80% in 8 h) was observed. Excision repair-deficient mutants generally cannot remove both CPD and 6–4 photoproducts. Though the radC mutant shows deficient excision repair, it can remove 6–4 photoproducts to a moderate degree. These results suggest that D. discoideum has two kinds of repair systems, one mainly for CPD and the other for 6–4 photoproducts, and that the radC mutant has a defect mainly in the repair enzyme for CPD.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Chemical investigation of light induced DNA bipyrimidine damage and repair

In all organisms, genetic information is stored in DNA and RNA. Both of these macromolecules are damaged by many exogenous and endogenous events, with UV irradiation being one of the major sources of damage. The major photolesions formed are the cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidone-(6-4)-photoproducts, Dewar valence isomers and, for dehydrated spore DNA, 5-(α-thyminyl)-5,6-dihydrothymine (SP). In order to be able to investigate how nature's repair and tolerance mechanisms protect the integrity of genetic information, oligonucleotides containing sequence and site-specific UV lesions are essential. This tutorial review provides an overview of synthetic procedures by which these oligonucleotides can be generated, either through phosphoramidite chemistry or direct irradiation of DNA. Moreover, a brief summary on their usage in analysing repair and tolerance processes as well as their biological effects is provided.     

Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in H^T7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200–280nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.     

DNA Ligases I and III Support Nucleotide Excision Repair in DT40 Cells with Similar Efficiency

In eukaryotic cells helix‐distorting DNA lesions like cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine‐pyrimidone photoproducts (6–4 PPs) are efficiently removed by nucleotide excision repair (NER). NER is a multistep process where in the end, subsequent to replication over the gap, the remaining nick is sealed by a DNA ligase. Lig1 has been implicated as the major DNA ligase in NER. Recently, Lig3 has been implicated as a component of a NER subpathway that operates in dividing cells, but which becomes particularly important in nondividing cells. Here, we use DT40 cells and powerful gene targeting approaches for generating DNA ligase mutants to examine the involvement and contribution of Lig1 and Lig3 in NER using cell survival measured by colony formation, and repair kinetics of CPD by immunofluorescence microscopy and immuno‐slot‐blotting. Our results demonstrate an impressive and previously undocumented potential of Lig3 to substitute for Lig1 in removing helix‐distorting DNA lesions by NER in proliferating cells. We show for the first time in a clean genetic background a functional redundancy in NER between Lig1 and Lig3, which appears to be cell cycle independent and which is likely to contribute to the stability of vertebrate genomes.     

Efficient Photoreactivation of Solar UV-Injured Metarhizium robertsii by Rad1 and Rad10 Linked to DNA Photorepair-Required Proteins

Nucleotide excision repair (NER) of ultraviolet (UV)-induced DNA lesions known as cyclobutane pyrimidine dimer (CPD) and (6–4)-pyrimidine-pyrimidone (6-4PP) photoproducts depends on the activities of multiple anti-UV radiation (RAD) proteins in budding yeast. However, NER remains poorly known in filamentous fungi, whose DNA lesions are photorepaired by one or two photolyases, namely CPD-specific Phr1 and/or 6-4PP-specific Phr2. Previously, the white collar proteins WC1 and WC2 were proven to regulate expressions of phr2 and phr1 and photorepair 6-4PP and CDP DNA lesions, respectively, in Metarhizium robertsii, a filamentous entomopathogenic-phytoendophytic fungus. We report here high activities of orthologous Rad1 and Rad10 in 5-h photoreactivation of UVB-injured or UVB-inactivated conidia but a severely compromised capability of their reactivating those conidia via 24-h dark incubation in M. robertsii. The null mutants of rad1 and rad10 were much more compromised in conidial UVB resistance and photoreactivation capability than the previous null mutants of phr1, phr2, wc1 and wc2. Multiple protein–protein (Rad1-Rad10, Rad1-WC2, Rad10-Phr1, WC1-Phr1/2 and WC2-Phr1/2) interactions detected suggest direct/indirect links of Rad1 and Rad10 to Phr1/2 and WC1/2 and an importance of the links for their photoreactivation activities. Conclusively, Rad1 and Rad10 photoreactivate UVB-impaired M. robertsii through their interactions with the DNA photorepair-required proteins.     

Decreased levels of (6-4) photoproduct excision repair in hybrid fish of the genus Xiphophorus

Selected hybridization in the fish genus Xiphophorus has been used for many years to study the genetics of malignant melanoma. Because DNA damage caused by ultraviolet radiation is implicated in the etiology of sunlight-induced melanoma, the heritability of mechanisms that mitigate DNA damage is a matter of some interest. We examined nucleotide excision repair of the two major types of DNA-damage induced by sunlight; the cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. In most cases, removal of the (6-4)PD was more rapid than the CPD, and in many cases, the F1 hybrid showed reduced repair efficiency compared with the parental species. These data demonstrate reduced function in multienzyme hybrid systems and provide molecular support for potential reduced fitness in hybrid fish under conditions of environmental stress.     

Long‐term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes

Photoprotection is essential to prevent the long‐term deleterious effects of ultraviolet (UV ), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE ) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA ‐induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA ‐irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE ‐treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.     

Repair of the main UV-induced thymine dimeric lesions within Arabidopsis thaliana DNA: evidence for the major involvement of photoreactivation pathways.

The UV-B induced formation of thymine cis-syn cyclobutane dimer and related (6-4) photoproduct was monitored within DNA of cultured cells and plants of Arabidopsis thaliana. This was achieved using a sensitive and accurate HPLC-tandem mass spectrometry assay. It was found that the cyclobutane pyrimidine dimer was formed in a ninefold higher yield than the (6-4) photoproduct. The removal of the lesions was then studied by incubating irradiated cells either in the darkness, under visible light or upon exposure to UV-A radiation. Dark repair of both cyclobutane dimers and (6-4) photoproducts was found to be very ineffective. In contrast, a rapid decrease in the level of photoproducts was observed when UV-B-irradiated cells were exposed to UV-A and, to a lesser extent, to visible light. The removal of (6-4) adducts was found to occur more efficiently. These results strongly suggest that repair of UV-induced photolesions in plants is mainly mediated by photolyases.     

Wavelength- and Tissue-dependent Variations in the Mutagenicity of Cyclobutane Pyrimidine Dimers in Mouse Skin

The cyclobutane pyrimidine dimer (CPD) is a main mutagenic photolesion in DNA produced by UVR. We previously studied the wavelength-dependent kinetics of mutation induction efficiency using monochromatic UVR sources and transgenic mice developed for mutation assay and established the action spectra of UVR mutagenicity in the mouse epidermis and dermis. Here, we further established the action spectra of CPD and pyrimidine(6-4)pyrimidone photoproduct formation in the same tissues and in naked DNA using the same sources and mouse strain. Quantitative ELISA helped us estimate the photolesion formation efficiencies on a molecule-per-nucleotide basis. Using these action spectra, we confirmed that the UVR mutation mostly depends on CPD formation. Moreover, the mutagenicity of a CPD molecule (CPD mutagenicity) was found to vary by wavelength, peaking at approximately 313 nm in both the epidermis and dermis with similar wavelength-dependent patterns. Thus, the CPD formation efficiency is a main determinant of UVR mutagenicity in mouse skin, whereas a wavelength-dependent variation in the qualitative characteristics of CPD molecules also affects the mutagenic consequences of UVR insults. In addition, the CPD mutagenicity was always higher in the epidermis than in the dermis, suggesting different cellular responses to UVR between the two tissues irrespective of the wavelength.