PostExcision Events in Human Nucleotide Excision Repair

The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage‐containing DNA oligonucleotide and a single‐stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single‐stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less‐explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.     

Formation and Direct Repair of UV‐induced Dimeric DNA Pyrimidine Lesions

Direct repair of UV‐induced DNA lesions represents an elegant method for many organisms to deal with these highly mutagenic and cytotoxic compounds. Although the participating proteins are structurally well investigated, the exact repair mechanism of the photolyase enzymes remains a vivid subject of current research. In this review, we summarize and highlight the recent contributions to this exciting field.     

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.     

Impact of the Circadian Clock on UV‐Induced DNA Damage Response and Photocarcinogenesis

The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time‐keeping mechanism, through regulation of the rate‐limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV‐induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock–NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.     

Deciphering UV-induced DNA Damage Responses to Prevent and Treat Skin Cancer

Ultraviolet (UV) radiation is among the most prevalent environmental factors that influence human health and disease. Even 1 h of UV irradiation extensively damages the genome. To cope with resulting deleterious DNA lesions, cells activate a multitude of DNA damage response pathways, including DNA repair. Strikingly, UV-induced DNA damage formation and repair are affected by chromatin state. When cells enter S phase with these lesions, a distinct mutation signature is created via error-prone translesion synthesis. Chronic UV exposure leads to high mutation burden in skin and consequently the development of skin cancer, the most common cancer in the United States. Intriguingly, UV-induced oxidative stress has opposing effects on carcinogenesis. Elucidating the molecular mechanisms of UV-induced DNA damage responses will be useful for preventing and treating skin cancer with greater precision. Excitingly, recent studies have uncovered substantial depth of novel findings regarding the molecular and cellular consequences of UV irradiation. In this review, we will discuss updated mechanisms of UV-induced DNA damage responses including the ATR pathway, which maintains genome integrity following UV irradiation. We will also present current strategies for preventing and treating nonmelanoma skin cancer, including ATR pathway inhibition for prevention and photodynamic therapy for treatment.     

Molecular assessment of UVC radiation-induced DNA damage repair in the stromatolitic halophilic archaeon, Halococcus hamelinensis

The halophilic archaeon Halococcus hamelinensis was isolated from living stromatolites in Shark Bay, Western Australia, that are known to be exposed to extreme conditions of salinity, desiccation, and UV radiation. Modern stromatolites are considered analogues of very early life on Earth and thus inhabitants of modern stromatolites, and Hcc. hamelinensis in particular, are excellent candidates to examine responses to high UV radiation. This organism was exposed to high dosages (up to 500 J/m(2)) of standard germicidal UVC (254 nm) radiation and overall responses such as survival, thymine-thymine cyclobutane pyrimidine dimer formation, and DNA repair have been assessed. Results show that Hcc. hamelinensis is able to survive high UVC radiation dosages and that intact cells give an increased level of DNA protection over purified DNA. The organism was screened for the bacterial-like nucleotide excision repair (NER) genes uvrA, uvrB, uvrC, as well as for the photolyase phr2 gene. All four genes were discovered and changes in the expression levels of those genes during repair in either light or dark were investigated by means of quantitative Real-Time (qRT) PCR. The data obtained and presented in this study show that the uvrA, uvrB, and uvrC genes were up-regulated during both repair conditions. The photolyase phr2 was not induced during dark repair, yet showed a 20-fold increase during repair in light conditions. The data presented is the first molecular study of different repair mechanisms in the genus Halococcus following exposure to high UVC radiation levels.     

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.     

Molecular Regulation of UV‐Induced DNA Repair

Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV‐induced DNA repair. When left unrepaired, UVB‐induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors.     

Proanthocyanidins from Grape Seeds Inhibit UV–Radiation‐Induced Immune Suppression in Mice: Detection and Analysis of Molecular and Cellular Targets

Ultraviolet (UV)–radiation‐induced immunosuppression has been linked with the risk of skin carcinogenesis. Approximately, 2 million new cases of skin cancers, including melanoma and nonmelanoma, diagnosed each year in the USA and therefore have a tremendous bad impact on public health. Dietary phytochemicals are promising options for the development of effective strategy for the prevention of photodamaging effects of UV radiation including the risk of skin cancer. Grape seed proanthocyanidins (GSPs) are such phytochemicals. Dietary administration of GSPs with AIN76A control diet significantly inhibits UV‐induced skin tumor development as well as suppression of immune system. UV‐induced suppression of immune system is commonly determined using contact hypersensitivity (CHS) model which is a prototype of T–cell‐mediated immune response. We present evidence that inhibition of UV‐induced suppression of immune system by GSPs is mediated through: (i) the alterations in immunoregulatory cytokines, interleukin (IL)‐10 and IL‐12, (ii) DNA repair, (iii) stimulation of effector T cells and (iv) DNA repair‐dependent functional activation of dendritic cells in mouse model. These information have important implications for the use of GSPs as a dietary supplement in chemoprevention of UV‐induced immunosuppression as well as photocarcinogenesis.     

Simultaneous Irradiation with Different Wavelengths of Ultraviolet Light has Synergistic Bactericidal Effect on Vibrio parahaemolyticus

Ultraviolet (UV) irradiation is an increasingly used method of water disinfection. UV rays can be classified by wavelength into UVA (320–400 nm), UVB (280‐320 nm), and UVC (<280 nm). We previously developed UVA sterilization equipment with a UVA light‐emitting diode (LED). The aim of this study was to establish a new water disinfection procedure using the combined irradiation of the UVA‐LED and another UV wavelength. An oxidative DNA product, 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG), increased after irradiation by UVA‐LED alone, and the level of cyclobutane pyrimidine dimers (CPDs) was increased by UVC alone in Vibrio parahaemolyticus. Although sequential irradiation of UVA‐LED and UVC‐induced additional bactericidal effects, simultaneous irradiation with UVA‐LED and UVC‐induced bactericidal synergistic effects. The 8‐OHdG and CPDs production showed no differences between sequential and simultaneous irradiation. Interestingly, the recovery of CPDs was delayed by simultaneous irradiation. The synergistic effect was absent in SOS response‐deficient mutants, such as the recA and lexA strains. Because recA‐ and lexA‐mediated SOS responses have crucial roles in a DNA repair pathway, the synergistic bactericidal effect produced by the simultaneous irradiation could depend on the suppression of the CPDs repair. The simultaneous irradiation of UVA‐LED and UVC is a candidate new procedure for effective water disinfection.     

Inflammation Due to Voriconazole‐induced Photosensitivity Enhanced Skin Phototumorigenesis in Xpa‐knockout Mice

Voriconazole is an antifungal agent and used as a prophylactic measure, especially in immunocompromised patients. However, there have been several reports of its adverse reactions, namely photosensitivity with intense inflammatory rashes and subsequent skin cancer development. To assess the effects of photosensitizing drugs voriconazole and hydrochlorothiazide (HCTZ ) on the enhancement of UV ‐induced inflammatory responses and UV ‐induced tumorigenesis, we utilized Xpa ‐knockout mice, which is DNA repair‐deficient and more susceptible to UV ‐induced inflammation and tumor development than wild‐type mice. Administration of voriconazole prior to broadband UVB exposure significantly upregulated multiple inflammatory cytokines compared with the vehicle‐ or HCTZ ‐administered groups. Voriconazole administration along with chronic UVB exposure produced significantly higher number of skin tumors than HCTZ or vehicle in Xpa ‐knockout mice. Furthermore, the investigation of UVB ‐induced DNA damage using embryonic fibroblasts of Xpa ‐knockout mice revealed a significantly higher 8‐oxo‐7,8‐dihydroguanine level in cells treated with voriconazole N‐oxide, a voriconazole‐metabolite during UV exposure. The data suggest that voriconazole plus UVB ‐induced inflammatory response may be related to voriconazole‐induced skin phototumorigenesis.     

Laser Microbeams and Optical Tweezers in Ageing Research

We show how a technique developed within the framework of physics and physical chemistry—in a true interdisciplinary approach—can answer questions in life sciences that are not solvable by using other techniques. Herein, we focus on blood‐pressure regulation and DNA repair in ageing studies. Laser microbeams and optical tweezers are now established tools in many fields of science, particularly in the life sciences. A short glimpse is given on the wide field of non‐age‐research applications in life sciences. Then, optical tweezers are used to show that exerting a vertical pressure on cells representing the inner lining of blood vessels results in bursts of NO liberation concomitant with large changes in cell morphology. Repeated treatment of such human umbilical vein endothelial cells (HUVEC) results in stiffening, a hallmark of manifest high blood pressure, a disease primarily of the elderly. As a second application in ageing research, a laser microbeam is used to induce, with high spatial and temporal resolution, DNA damages in the nuclei of U2OS human osteosarcoma cells. A pairwise study of the recruitment kinetics of different DNA repair proteins reveals that DNA repair starts with non‐homologous end joining (NHEJ), a repair pathway, and may only after several minutes switch to the error‐free homologous recombination repair (HRR) pathway. Since DNA damages—when incorrectly repaired—accumulate with time, laser microbeams are becoming well‐used tools in ageing research.     

Autophagy in UV Damage Response

UV radiation exposure from sunlight and artificial tanning beds is the major risk factor for the development of skin cancer and skin photoaging. UV‐induced skin damage can trigger a cascade of DNA damage response signaling pathways, including cell cycle arrest, DNA repair and, if damage is irreparable, apoptosis. Compensatory proliferation replaces the apoptotic cells to maintain skin barrier integrity. Disruption of these processes can be exploited to promote carcinogenesis by allowing the survival and proliferation of damaged cells. UV radiation also induces autophagy, a catabolic process that clears unwanted or damaged proteins, lipids and organelles. The mechanisms by which autophagy is activated following UV exposure, and the functions of autophagy in UV response, are only now being clarified. Here, we summarize the current understanding of the mechanisms governing autophagy regulation by UV, the roles of autophagy in regulating cellular response to UV‐induced photodamage and the implications of autophagy modulation in the treatment and prevention of photoaging and skin cancer.     

Hormonal Regulation of the Repair of UV Photoproducts in Melanocytes by the Melanocortin Signaling Axis

Melanoma is the deadliest form of skin cancer because of its propensity to spread beyond the primary site of disease and because it resists many forms of treatment. Incidence of melanoma has been increasing for decades. Although ultraviolet radiation (UV) has been identified as the most important environmental causative factor for melanoma development, UV‐protective strategies have had limited efficacy in melanoma prevention. UV mutational burden correlates with melanoma development and tumor progression, underscoring the importance of UV in melanomagenesis. However, besides amount of UV exposure, melanocyte UV mutational load is influenced by the robustness of nucleotide excision repair, the genome maintenance pathway charged with removing UV photoproducts before they cause permanent mutations in the genome. In this review, we highlight the importance of the melanocortin hormonal signaling axis on regulating efficiency of nucleotide excision repair in melanocytes. By understanding the molecular mechanisms by which nucleotide excision repair can be increased, it may be possible to prevent many cases of melanoma by reducing UV mutational burden over time.     

Beyond Xeroderma Pigmentosum: DNA Damage and Repair in an Ecological Context. A Tribute to James E. Cleaver

The ability to repair DNA is a ubiquitous characteristic of life on Earth and all organisms possess similar mechanisms for dealing with DNA damage, an indication of a very early evolutionary origin for repair processes. James E. Cleaver's career (initiated in the early 1960s) has been devoted to the study of mammalian ultraviolet radiation (UVR) photobiology, specifically the molecular genetics of xeroderma pigmentosum and other human diseases caused by defects in DNA damage recognition and repair. This work by Jim and others has influenced the study of DNA damage and repair in a variety of taxa. Today, the field of DNA repair is enhancing our understanding of not only how to treat and prevent human disease, but is providing insights on the evolutionary history of life on Earth and how natural populations are coping with UVR‐induced DNA damage from anthropogenic changes in the environment such as ozone depletion.     

MOLECULAR CLONING OF THE HUMAN GENE SUVCC1 ASSOCIATED WITH THE REPAIR OF NONDIMER DNA DAMAGE INDUCED BY SOLAR UV RADIATION

Abstract— A mutant cell line, DRP 287, sensitive to solar UV radiation and deficient in the repair of solar UV-induced nondimer DNA damage, was derived from ICR 2A frog cells. These cells were transfected with human DNA and a secondary transformant obtained in which normal solar UV sensitivity was restored and the repair defect corrected. The DNA from this secondary transformant was used to construct a genomic DNA library from which a recombinant phage was isolated containing the human gene capable of restoring normal solar UV sensitivity and correcting the repair defect in the DRP 287 cells. This represents the first human gene which has been isolated that is specifically involved in the repair of nondimer DNA damage induced by solar UV radiation. It has been designated SUVCC1 to denote solar UV cross-complementing gene number 1.     

UV Radiation Increases Carcinogenic Risks for Oral Tissues Compared to Skin

People can expose their oral cavities to UV (290–400 nm) by simply opening their mouths while outdoors. They can also have their oral cavities exposed to UV indoors to different UV‐emitting devices used for diagnoses, treatments and procedures like teeth whitening. Because the World Health Organization declared UV radiation as a complete human carcinogen in 2009, we asked if oral tissues are at a similar or higher carcinogenic risk compared to skin tissue. To understand the UVB (290–320 nm)‐related carcinogenic risks to these tissues, we measured initial DNA damage in the form of cyclobutane pyrimidine dimers (CPD), the repair rate of CPD (24 h) and the number of apoptotic dead cells over time resulting from increasing doses of erythemally weighted UV radiation. We used commercially available 3D‐engineered models of human skin (EpiDerm?), gingival (EpiGingival?) and oral (EpiOral?) tissues and developed an analytical approach for our tri‐labeling fluorescent procedure to identify total DNA, CPD and apoptotic cells so we can simultaneously quantify DNA repair rates and dead cells. Both DNA repair and apoptotic cell numbers are significantly lower in oral cells compared with skin cells. The combined results suggest UVB‐exposed oral tissues are at a significantly higher carcinogenic risk than skin tissues.