Insight in DNA Repair of UV‐induced Pyrimidine Dimers by Chromatographic Methods

UV‐induced formation of pyrimidine dimers in DNA is a major deleterious event in both eukaryotic and prokaryotic cells. Accumulation of cyclobutane pyrimidine dimers and pyrimidine (6‐4) pyrimidone photoproducts can lead to cell death or be at the origin of mutations. In skin, UV induction of DNA damage is a major initiating event in tumorigenesis. To counteract these deleterious effects, all cell types possess DNA repair machinery, such as nucleotide excision repair and, in some cell types, direct reversion. Different analytical approaches were used to assess the efficiency of repair and decipher the enzymatic mechanisms. We presently review the information provided by chromatographic methods, which are complementary to biochemical assays, such as immunological detection and electrophoresis‐based techniques. Chromatographic assays are interesting in their ability to provide quantitative data on a wide range of damage and are also valuable tools for the identification of repair intermediates.     

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.     

Bringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint

Nucleotide excision repair and the ATR‐mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al. developed an in vitro coupled repair‐checkpoint system to analyze the basic steps of these DNA damage stress responses in a biochemically defined system. The minimum set of factors essential for repair‐checkpoint coupling include damaged DNA, the excision repair factors (XPA, XPC, XPF‐ERCC1, XPG, TFIIH, RPA), the 5′‐3′ exonuclease EXO1, and the damage checkpoint proteins ATR‐ATRIP and TopBP1. This coupled repair‐checkpoint system was used to demonstrate that the ~30 nucleotide single‐stranded DNA (ssDNA) gap generated by nucleotide excision repair is enlarged by EXO1 and bound by RPA to generate the signal that activates ATR.     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6‐4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair‐deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair‐proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

THE EFFECT OF TEMPERATURE AND WAVELENGTH ON PRODUCTION AND PHOTOLYSIS OF A UV-INDUCED PHOTOSENSITIVE DNA LESION WHICH IS NOT REPAIRED IN XERODERMA PIGMENTOSUM VARIANT CELLS

Abstract— Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0^°C and 37^°C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0^°C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and(6–4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine(6–4) photoproducts     

Quantitative and in situ Detection of Oxidatively Generated DNA Damage 8,5′‐Cyclo‐2′‐Deoxyadenosine Using an Immunoassay with a Novel Monoclonal Antibody

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix‐distorting types of DNA damage including sunlight‐induced pyrimidine dimers. In addition to skin disease, approximately 30% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of a particular type of oxidatively generated DNA damage called purine 8,5′‐cyclo‐2′‐deoxynucleosides (purine cyclonucleosides). However, there are no currently available methods to detect purine cyclonucleosides in DNA without the need for DNA hydrolysis. In this study, we generated a novel monoclonal antibody (CdA‐1) specific for purine cyclonucleosides in single‐stranded DNA that recognizes 8,5′‐cyclo‐2′‐deoxyadenosine (cyclo‐dA). An immunoassay using CdA‐1 revealed a linear dose response between known amounts of cyclo‐dA in oligonucleotides and the antibody binding to them. The quantitative immunoassay revealed that treatment with Fenton‐type reagents (CuCl₂/H₂O₂/ascorbate) efficiently produces cyclo‐dA in DNA in a dose‐dependent manner. Moreover, immunofluorescent analysis using CdA‐1 enabled the visualization of cyclo‐dA in human osteosarcoma cells, which had been transfected with oligonucleotides containing cyclo‐dA. Thus, the CdA‐1 antibody is a valuable tool for the detection and quantification of cyclo‐dA in DNA, and may be useful for characterizing the mechanism(s) underlying the development of XP neurological disease.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Identification and genetic characterization of Anisakis larvae from marine fishes in the South China Sea using an electrophoretic‐guided approach

From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR‐based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3′‐end of the small subunit and 5′‐end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA‐2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC‐2005; originating from the African shelf) and another sequence (PH‐2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA‐2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA‐2012 represents a new record, now called Anisakis type III larvae.