Repair Of UV-induced DNA Damage Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Divergence of cAMP signalling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes.

Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signalling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signalling positions melanocytes to resist UV injury by upregulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia-mutated and Rad3-related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signalling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated upregulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (eg PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (eg MITF activation) are distinct.

752 Phosphorylation of XPC regulates XPC’s function in UV-induced DNA damage repair

752 Phosphorylation of XPC regulates XPC’s function in UV-induced DNA damage repair

DGCR8 Mediates Repair of UV-Induced DNA Damage Independently of RNA Processing

Ultraviolet (UV) radiation is a carcinogen that generates DNA lesions. Here, we demonstrate an unexpected role for DGCR8, an RNA binding protein that canonically functions with Drosha to mediate microRNA processing, in the repair of UV-induced DNA lesions. Treatment with UV induced phosphorylation on serine 153 (S153) of DGCR8 in both human and murine cells. S153 phosphorylation was critical for cellular resistance to UV, the removal of UV-induced DNA lesions, and the recovery of RNA synthesis afterUV exposure but not for microRNA expression. The RNA-binding and Drosha-binding activities of DGCR8 were not critical for UV resistance. DGCR8 depletion was epistatic to defects in XPA, CSA, and CSB for UV sensitivity. DGCR8 physically interacted with CSB and RNA polymerase II. JNKs were involved in the UV-induced S153 phosphorylation. These findings suggest that UV-induced S153 phosphorylation mediates transcription-coupled nucleotide excision repair of UV-induced DNA lesions in a manner independent of microRNA processing.

Ultraviolet radiation: DNA damage, repair, and human disorders

Ultraviolet (UV) radiation is one of the most common environmental health hazards that cause highly toxic effects in most living organisms. UV irradiation leads to harmful effects including skin aging, eye damage, and skin cancer because of increased production of cellular reactive oxygen species and by direct DNA damage. Damaged DNA, if not properly repaired, is a source of mutation, and interferes with many cellular mechanisms such as replication, transcription, and the cell cycle. Because most UV damaged DNA is efficiently repaired by nucleotide excision repair (NER), which is a specialized UV-induced DNA damage repair system, many UV-induced symptoms are closely related to NER. Therefore, understanding the function of NER genes will elucidate the cause of different UV-induced symptoms. Furthermore, a multidisciplinary understanding of damaged DNA repair systems and other cellular mechanisms affected by unrepaired DNA damage would lead to an improved understanding of UV-induced symptoms and toward developing various preventive and therapeutic methods against UV damage. For this purpose, in this review we discuss two NER-related human genetic disorders, xeroderma pigmentosum (XP) and Cockayne syndrome (CS), the cellular mechanisms that are impaired by defective NER genes, and the functions of RAD2/XPG in relation to the cause of various UV damage-induced symptoms.

Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage.

UVC irradiation-caused DNA lesions are repaired in mammalian cells solely by nucleotide excision repair (NER), which consists of sequential events including initial damage recognition, dual incision of damage site, gap-filling, and ligation. We have previously shown that gap-filling during the repair of UV-induced DNA lesions may be delayed by a subsequent treatment of oxidants or prooxidants such as hydrogen peroxide, flavonoids, and colcemid. We considered the delay as a result of competition for limiting protein/enzyme factor(s) during repair synthesis between NER and base excision repair (BER) induced by the oxidative chemicals. In this report, using colcemid as oxidative stress inducer, we showed that colcemid-caused delay of gap-filling during the repair of UV-induced DNA lesions was attenuated by overexpression of PCNA but not ligase-I. PCNA knockdown, as expected, delayed the gap-filling of NER but also impaired the repair of oxidative DNA damage. Fen-1 knockdown, however, did not affect the repair of oxidative DNA damage, suggesting repair of oxidative DNA damage is not of long patch BER. Furthermore, overexpression of XRCC1 delayed the gap-filling, and presumably increase of XRCC1 pulls PCNA away from gap-filling of NER for BER, consistent with our hypothesis that delay of gap-filling of NER attributes the competition between NER and BER.

Impact of UV Radiation on Genome Stability and Human Health.

Gradual depletion of the atmospheric ozone layer during the past few years has increased the incidence of solar UV radiation specifically the UV-C on earth's surface is one of the major environmental concerns because of the harmful effects of this radiation in all forms of life. The solar UV radiation including the harmful wavelength range of UV-B (280-320nm) represents a significant climatic stress for both animals and plants, causing damage to the fundamental biomolecules such as DNA, proteins and lipids, thus activating genotoxic stress and induces genome instability. When DNA absorbs UV-B light, energy from the photon causes covalent linkages to form between adjacent pyrimidine bases, creating photoproducts, primarily cyclobutane pyrimidine dimers (CPDs) and pyrimidine-6,4-pyrimidinone photoproduct (6,4PPs). Pyrimidine dimers create distortions in the DNA strands and therefore can inhibit DNA replication as well transcription. Lack of efficient repair of UV-induced DNA damage may induce the formation of DNA double stand breaks (DSBs), one of the serious forms of damage in DNA double helix, as well as oxidative damage. Unrepaired DSBs in the actively dividing somatic cells severely affect cell growth and development, finally results in loss of cell viability and development of various diseases, such as cancer in man.This chapter mainly highlights the incidence of solar UV-radiation on earth's surface along with the formation of major types of UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and finally our present understanding on the impact on solar UV radiation on human health.

The DNA-binding box of human SPARTAN contributes to the targeting of Polη to DNA damage sites

Inappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an error-free manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNA-binding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function.

Autophagic UVRAG Promotes UV-Induced Photolesion Repair by Activation of the CRL4DDB2 E3 Ligase

UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover toXPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.

MiR-183 Inhibits UV-Induced DNA Damage Repair in Human Trabecular Meshwork Cells by Targeting of KIAA0101.

PurposeThe purpose of this study was to investigate the mechanisms by which miR-183 may contribute to the phenotypic alterations associated with stress-induced senescence of human trabecular meshwork (HTM) cells.MethodsChanges in gene expression induced by miR-183 in HTM cells were evaluated by gene array analysis, confirmed by quantitative-PCR (Q-PCR), and analyzed by MetaCore pathway analysis. Effects of miR-183 on cell proliferation were assessed by incorporation of bromodeoxyuridine incorporation, and DNA damage by CometAssay after ultraviolet (UV) irradiation in primary HTM cells, and confirmed in human diploid fibroblasts (HDF) and HeLa cells. A plasmid expressing KIAA0101 without its 3′-untranslated region (3′-UTR) was cotransfected with miR-183 to evaluate the role of KIAA0101 on the effects induced by miR-183.ResultsmiR-183 affected the expression of multiple genes involved in cell cycle regulation and DNA damage response in HTM cells. Forced expression of miR-183 in HTM and HDF resulted in a significant decrease in proliferation in primary HTM and HDF cells but not in HeLa cells. In all cell types tested, overexpression of miR-183 resulted in increased DNA damage under UV irradiation. Expression of KIAA0101 lacking the 3′-UTR region partially prevented the effects of miR-183 on cell proliferation and completely reversed the effects on UV-induced DNA damage.ConclusionsOur results suggest that the observed up-regulation of miR-183 after stress-induced senescence in HTM cells may contribute to reinforce cellular senescence by inhibiting cell cycle progression through multiple gene targets and limiting the DNA repair mechanisms through inhibition of KIAA0101.

608 Targeting the MC1R by selective tetra- and tripeptide analogs of α-Melanocortin for melanoma prevention

Our goal is to develop a topical formulation of small peptide analogs of α-Melanocortin (α-melanocyte stimulating hormone; α-MSH) that are highly selective for the melanocortin 1 receptor (MC1R) for prevention of photodamage in human melanocytes (HM) and ultimately melanoma. The MC1R is a melanoma susceptibility gene, and its loss-of-function variants result in red hair phenotype, reduced tanning and DNA repair capacity, and increased melanoma risk. Activation of the MC1R by its physiological agonist α-MSH stimulates eumelanin synthesis and proliferation, enhances the repair of UV-induced DNA damage and inhibits apoptosis of HM. We have designed short peptide analogs of the physiological tridecapeptide α-MSH exhibiting full MC1R agonism and excellent MC1R selectivity. Modifications of the His-D-Phe-Arg-Trp, or His-D-Phe-Arg sequence of α-MSH combined with terminal modifications resulted in stable small molecules with logD favorable for skin penetration. The tetrapeptides LK 184 and LK 467, and the tripeptide LK 514 were tested on primary cultures of HM and cultured skin substitutes (CSS). Dose-response experiments revealed that LK 184 and LK 467 were 100 fold more potent, while LK 514 was slightly less (≤10 fold) potent than α-MSH in stimulating cAMP formation and the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, in HM. The three peptides enhanced the repair of UV-induced DNA damage in HM. Treatment of CSS with LK 184 and LK 467 at 100 nM, or LK 514 at -5 μM for 10-15 days significantly increased pigmentation in the absence of UV exposure, and increased repair of UV-induced DNA photoproducts in melanocytes and the epidermis in the CSS or intact human skin. Prolonged treatment with these peptides had no effect on melanocyte number or histology of CSS. Given that increased pigmentation and DNA repair protect against photocarcinogenesis, we are continuing with preclinical testing of these peptides to determine their safety and efficacy as topical agents for sunless tanning and prevention of photodamage and melanoma.

574 Ubiquitin-specific peptidase 11 regulates XPC activity in UV-induced DNA damage repair

574 Ubiquitin-specific peptidase 11 regulates XPC activity in UV-induced DNA damage repair

UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis.

The COP9/signalosome (CSN) multi-protein complex regulates the activity of cullin-RING ubiquitin ligases (CRLs), including the DDB2 and CSA CRL4 ligases (CRL4DDB2 and CRL4CSA), which are involved in the repair of UV-induced DNA damages. In the present study, we demonstrated that the protein kinase ATM, a key component of the DNA damage response (DDR), phosphorylates CSN1 and CSN7a, two subunits of the CSN complex, in a UV-dependent manner. The phosphorylation of CSN1 on serine 474 was detected as early as 3 h after UV-exposure, peaked at 8 h and persisted until 48 h post-UV irradiation. Such a time course suggests a role in late DDR rather than in DNA repair. Consistently, overexpression of a phosphorylation-resistant S474A CSN1 mutant reduced UV-induced apoptosis. Thus, CSN1 appears to play a role not only in DNA repair but also in UV-induced apoptosis.

XPG rs2296147 T>C polymorphism predicted clinical outcome in colorectal cancer.

Xeroderma pigmentosum group G (XPG), one of key components of nucleotide excision repair pathway (NER), is involved in excision repair of UV-induced DNA damage. Single nucleotide polymorphisms (SNPs) in the XPG gene have been reported to associate with the clinical outcome of various cancer patients. We aimed to assess the impact of four potentially functional SNPs (rs2094258 C>T, rs2296147 T>C, rs751402 G>A, and rs873601 G>A) in the XPG gene on prognosis in colorectal cancer (CRC) patients. A total of 1901 patients diagnosed with pathologically confirmed CRC were genotyped for four XPG polymorphisms. Cox proportional hazards model analysis controlled for several confounding factors was conducted to compute hazard ratios (HRs) and 95% confidence intervals (CIs). Of the four included SNPs, only rs2296147 was shown to significantly affect progression-free survival (PFS) in CRC. Patients carrying rs2296147 CT/TT genotype had a significantly shorter median 10 years PFS than those carrying CC genotype (88.5 months vs. 118.1 months), and an increased progression risk were observed with rs2296147 (HR = 1.324, 95% CI = 1.046–1.667). Moreover, none of the four SNPs were associated with overall survival. In conclusion, our study showed that XPG rs2296147 CT/TT variants conferred significant survival disadvantage in CRC patients in term of PFS. XPG rs2296147 polymorphism could be predictive of unfavorable prognosis of CRC patients.

Mitochondrial dysfunction activates the AMPK signaling and autophagy to promote cell survival

Autophagy is a cellular self-eating process essential for stress response and maintaining tissue homeostasis by lysosomal degradation of unwanted or damaged proteins and organelles. Here, we show that cells with defective mitochondria induce autophagy to promote cell survival through activating the AMPK pathway. Loss of mitochondrial complex III protein cytochrome b activates the AMPK signaling and induced autophagy. Inhibiting mitochondria energetics by mitochondria-targeted agents activates the AMPK signaling and induced autophagy. Genetic inhibition of AMPK inhibits autophagy induction in cells with defective mitochondria, while genetic inhibition of autophagy has no effect on AMPK activation. Mitochondria dysfunction has no effect of DNA repair of UV-induced DNA damage. However, mitochondria dysfunction sensitizes cells to apoptosis induced by UV radiation. Genetic inhibition of autophagy or AMPK sensitized cells to apoptosis in cells with defective mitochondria. Our results demonstrate that AMPK and autophagy senses mitochondria dysfunction and serves as a mechanism for survival. Our findings may provide new insights into the interplay between mitochondria function and autophagy process in maintaining tissue homeostasis, and suggest that this interaction may play important roles in diseases such as cancer and neurodegeneration.

Beyond Red Hair and Sunburns: Uncovering the Molecular Mechanisms of MC1R Signaling and Repair of UV-Induced DNA Damage

Scientists at the University of Kentucky are unravelling the details of DNA damage repair in the melanocyte, with an eye towards finding druggable targets for melanoma prevention. Jarret et al. report in this issue three new assays that can yield mechanistic information about nucleotide excision repair (NER) stimulated by cAMP-dependent signaling downstream of the melanocortin-1 receptor (MC1R).

TGF-β signaling links E-cadherin loss to suppression of nucleotide excision repair.

E-cadherin is a cell adhesion molecule best known for its function in suppressing tumor progression and metastasis. Here we show that E-cadherin promotes nucleotide excision repair through positively regulating the expression of xeroderma pigmentosum complementation group C (XPC) and DNA damage-binding protein 1 (DDB1). Loss of E-cadherin activates the E2F4 and p130/107 transcription repressor complexes to suppress the transcription of both XPC and DDB1 through activating the TGF-β pathway. Adding XPC or DDB1, or inhibiting the TGF-β pathway, increases the repair of UV-induced DNA damage in E-cadherin-inhibited cells. In mouse skin and skin tumors UVB radiation down-regulates E-cadherin. In sun-associated premalignant and malignant skin neoplasia, E-cadherin is down-regulated in association with reduced XPC and DDB1 levels. These findings demonstrate a crucial role of E-cadherin in efficient DNA repair of UV-induced DNA damage, identify a new link between epithelial adhesion and DNA repair, and suggest a mechanistic link of early E-cadherin loss in tumor initiation.

Chromatin perturbations during the DNA damage response in higher eukaryotes.

The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.

The tomato DDI2, a PCNA ortholog, associating with DDB1-CUL4 complex is required for UV-damaged DNA repair and plant tolerance to UV stress

CULLIN 4 (CUL4)-DAMAGED DNA binding protein 1 (DDB1)-based ubiquitin E3 ligase modulates diverse cellular processes including repair of damaged genomic DNA. In this study, an uncharacterized gene termed as DDB1-Interacting protein 2 (DDI2) was identified in yeast two-hybrid screening with bait gene DDB1. The co-immunoprecipitation (co-IP) assays further demonstrated that DDI2 is associated with tomato DDB1-CUL4 complex in vivo. It appears that DDI2 encodes an ortholog of proliferating cell nuclear antigen (PCNA). Confocal microscope observation indicated that DDI2-GFP fusion protein was localized in nuclei. The expression of DDI2 gene is constitutive but substantially enhanced by UV-C irradiation. The transgenic tomato plants with overexpression or knockdown of DDI2 gene displayed the increased or decreased tolerance, respectively, to UV-C stress and chemical mutagen cisplatin. The quantitative analysis of UV-induced DNA lesions indicated that the dark repair of DNA damage was accelerated in DDI2 overexpression lines but delayed in knockdown lines. Conclusively, tomato DDI2 gene is required for UV-induced DNA damage repair and plant tolerance to UV stress. In addition, fruits of DDI2 transgenic plants are indistinguishable from that of wild type, regarding fresh weight and nutrient quality. Therefore, overexpression of DDI2 offers a suitable strategy for genetic manipulation of enhancing plant tolerance to UV stress.

Overexpression of Bm65 correlates with reduced susceptibility to inactivation by UV light

Ultraviolet (UV) light is one of the factors that causes baculovirus inactivation. However, little is known about the response of baculoviruses to UV light. In the present study, Bombyx mori nucleopolyhedrovirus (BmNPV) orf 65 (Bm65), the homolog of Autographa californica nucleopolyhedrovirus orf 79 (Ac79), a predicted endonuclease, was analyzed. Preliminary results indicated that Bm65 mainly accumulated within the nucleus and could improve the survival rate of Escherichia coli (E. coli) and BmNPV BVs after UV radiation, suggesting that Bm65 was involved in the repair of UV-induced DNA damage.