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.     

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 cytosine methylation on pyrimidine dimer formation in DNA

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)pyrimidone photoproducts ([6-4]PD) was quantified in the duplex homopolymers polydeoxyadenosine:polydeoxythymidine, polydeoxyguanosine:polydeoxycytidine and polydeoxyguanosine:polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6-4)PD after irradiation with UVC light and of cytosine CPD and (6-4)PD after irradiation with UVB light. The data suggest that CPD and (6-4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.     

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.     

Diel Cycles of DNA Damage and Repair in Eggs and Larvae of Northern Anchovy, Engraulis mordax, Exposed to Solar Ultraviolet Radiation

Abstract— Damage from UVB radiation (280–320 nm) in the form of cyclobutane pyrimidine dimers (CPD) in DNA and the capacity for their repair were measured in newly spawned eggs and yolk-sac larvae of northern anchovy, Engraulis mordax, exposed to natural diel cycles of sunlight. The CPD were measured by a newly developed chemiluminescent immunoblot assay capable of measuring CPD in samples as small as 50 ng DNA. Eggs and yolk-sac larvae exposed to full irradiance levels died. At lower dose rates, equivalent to deeper more natural locations in the water column, there was a diel cycle of dimer concentration that tracked solar intensity. This diel cycle was due to the interaction of damage and repair processes. Repair of CPD in anchovy eggs and larvae could be attributed to true photodependent repair that could be stopped by moving samples into the dark. The CPD present at sunset remained until the following morning. The diel cycles of damage and repair were maintained over at least 4 days without a long-term upward or downward trend in dimer concentration. This indicates that at the UVB doses used for these experiments, there was no long-term accumulation of CPD nor an induction of increased repair capacity. Unhatched embryos spawned in the dark also exhibited a strong photorepair response, suggesting that photolyase expression was innate and not dependent on previous light exposure. The diel cycle observed here indicates that, at least for northern anchovy, the CPD concentration at the time of sampling is a good indicator of dose rate but a poor indicator of cumulative dose (i.e. late afternoon samples have the highest cumulative dose but relatively low CPD concentrations). The CPD immunoassay described here has the required sensitivity for measuring DNA damage in wild populations of ichthyoplankton exposed to natural sunlight. These results will guide the collection and interpretation of field data on natural levels of CPD in wild larvae collected at different depths and times of the day.     

Computational studies of DNA photolyase

The electron transfer catalyzed (ETC) repair of the DNA photolesion cyclobutane pyrimidine dimer (CPD) is mediated by the enzyme DNA photolyase. Due to its importance as part of the cancer prevention mechanism in many organisms, but also due to its unique mechanism, this DNA photoreactivation is a topic of intense study. The progress in the application of computational methods to three aspects of the ETC repair of CPD is reviewed: (i) electronic structure calculations of the cycloreversion of the CPD radical cation and radical anion, (ii) MD simulations of the DNA photolyase and its complex to photodamaged DNA, and (iii) the structure and dynamics of photodamaged DNA. The contributions of this work to the overall understanding of the reaction and its relationship to the available experimental work are highlighted.     

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.     

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.     

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.     

Influence of Phage Proteins on Formation of Specific UV DMA Photoproducts in Phage T7

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.     

Establishment of a microplate-formatted cell-based immunoassay for rapid analysis of nucleotide excision repair ability in human primary cells

DNA photolesions induced by UV, cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP), are repaired by nucleotide excision repair (NER) in human cells. Various immunoassays using monoclonal antibodies specific for the photolesions have been developed and widely used for the analysis of cellular NER activity. In this study, we have newly developed a microplate-formatted cell-based immunoassay, based on indirect immunofluorescence staining with lesion-specific antibodies combined with an infrared imaging system. Using this assay, we show the repair kinetics of CPD and 6-4PP in various fibroblasts from newborn and adult donors with no age-related difference. Furthermore, epidermal keratinocytes and melanocytes exhibit comparable NER activity, and calcium ion-induced differentiation of keratinocytes has no significant impacts on their NER activity. We also evaluated the effects of a proteasome inhibitor, MG132, and a histone deacetylase inhibitor, sodium butyrate, on NER efficiency using this assay. All these results suggest that the new assay is highly useful for the rapid and quantitative analysis of NER activity in various primary cells with limited growth activity and is applicable to a screening system for drugs affecting NER efficiency.     

PREFERENTIAL INHIBITION OF NUCLEOSOME ASSEMBLY BY ULTRAVIOLET-INDUCED (6-4)PHOTOPRODUCTS

We reconstituted nucleosomes in vitro using two kinds of damaged pBR322 plasmid DNA carrying cyclobutane pyrimidine dimers (CPD) or (6-4)photoproducts. The results indicate that nucleosome assembly is inhibited preferentially by (6-4)photoproducts compared with CPD, suggesting that the regions carrying (6-4)photoproducts retain their nucleosome-free form, i.e. linker-like conformation until completion of the repair processes.     

Patterns of DNA damage and photoinhibition in temperate South-Atlantic picophytoplankton exposed to solar ultraviolet radiation.

Natural marine phytoplankton assemblages from Bahía Bustamante (Chubut, Argentina, 45 degrees S, 66.5 degrees W), mainly consisting of cells in the picoplankton size range (0.2-2 microm), were exposed to various UVBR (280-315 nm) and UVAR (315-400 nm) regimes in order to follow wavelength-dependent patterns of cyclobutane pyrimidine dimer (CPD) induction and repair. Simultaneously, UVR induced photosynthetic inhibition was studied in radiocarbon incorporation experiments. Biological weighting functions (BWFs) for photoinhibition and for CPD induction, the latter measured in bare calf thymus DNA, differed in the UVAR region: carbon incorporation was reduced markedly due to UVAR, whereas no measurable UVAR effect was found on CPD formation. In contrast, BWFs for inhibition of photosynthesis and CPD accumulation were fairly similar in the UVBR region, especially above 300 nm. Incubation of phytoplankton under full solar radiation caused rapid CPD accumulation over the day, giving maximum damage levels exceeding 500 CPD MB(-1) at the end of the afternoon. A clear daily pattern of CPD accumulation was found, in keeping with the DNA effective dose measured by a DNA dosimeter. In contrast, UVBR induced photosynthetic inhibition was not dose related and remained nearly constant during the day. Screening of UVBR or UVR did not cause significant CPD removal, indicating that photoreactivation either by PAR or UVAR was of minor importance in these organisms. High CPD levels were found in situ early in the morning, which remained unaffected notwithstanding treatments favoring photorepair. These results imply that a proportion of cells had been killed by UVBR exposure prior to the treatments. Our data suggest that the limited potential for photoreactivation in picophytoplankton assemblages from the southern Atlantic Ocean causes high CPD accumulation as a result of UVBR exposure.     

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.     

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.     

An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB‐induced Melanoma

Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB‐inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor‐bearing and tumor‐free fish were given a challenge UVB dose and (6–4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter‐individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter‐individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.     

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.     

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.     

Base flipping of the thymine dimer in duplex DNA

Exposure of two adjacent thymines in DNA to UV light of 260-320 nm can result in the formation of the cis,syn-cyclobutane pyrimidine dimer (CPD). The structure of DNA containing an intrahelical CPD lesion has been previously studied experimentally and computationally. However, the structure of the extrahelical, flipped-out, CPD lesion, which has been shown to be the structure that binds to the CPD repair enzyme, DNA photolyase, has yet to be reported. In this work the structure of both the flipped-in and the flipped-out CPD lesions in duplex DNA is reported. These structures were calculated using 8 ns molecular dynamics (MD) simulations. These structures are then used to define the starting and ending points for the base-flipping process for the CPD lesion. Using a complex, two-dimensional pseudodihedral coordinate, the potential of mean force (PMF) for the base-flipping process was calculcated using novel methodology. The free energy of the flipped-out CPD is roughly 6.5 kcal/mol higher than that of the flipped-in state, indicating that the barrier to flipping out is much lower for CPD than for undamaged DNA. This may indicate that the flipped-out CPD lesion may be recognized by its repair enzyme, DNA photolyase, whereas previous studies of other damaged, as well as nondamaged, bases indicate that they are recognized by enzymes in the intrahelical, flipped-in state.