Nucleotide Excision Repair System Research Articles

The REV-ERB antagonist SR8278 modulates keratinocyte viability in response to UVA and UVB radiation.

The nucleotide excision repair (NER) system removes UV photoproducts from genomic DNA and is controlled by the circadian clock. Given that small-molecule compounds have been developed to target various clock proteins, we examined whether the cryptochrome inhibitor KS15 and REV-ERB antagonist SR8278 could modulate keratinocyte responses to UV radiation invitro. We observed that though SR8278 promoted cell viability in UVB-irradiated cells, it had little effect on NER or on the expression of the clock-regulated NER factor XPA. Rather, we found that both KS15 and SR8278 absorb light within the UV spectrum to limit initial UV photoproduct formation in DNA. Moreover, SR8278 promoted UVB viability even in cells in which the core circadian clock protein BMAL1 was disrupted, which indicates that SR8278 is likely acting via other REV-ERB transcriptional targets. We further observed that SR8278 sensitized keratinocytes to light sources containing primarily UVA wavelengths of light likely due to the generation of toxic reactive oxygen species. Though other studies have demonstrated beneficial effects of SR8278 in other model systems, our results here suggest that SR8278 has limited utility for UV photoprotection in the skin and will likely cause phototoxicity in humans or mammals exposed to solar radiation.

Nucleotide excision repair. Methods of determination the efficiency functioning

The nucleotide excision repair (NER) system is responsible for removing a wide range of bulky damages from DNA, making a significant contribution to maintaining the stability of the genome. The efficiency with which the proteins of the NER system recognize and remove bulky damages depends on many factors and has important clinical and diagnostic significance. The review examines the current understanding of the molecular basis of the functioning of the NER system in eukaryotic cells, as well as analyzes the methods and approaches that are used to study the efficiency functioning of this DNA repair system both in vitro and ex vivo.

Methods for Assessment of Nucleotide Excision Repair Efficiency.

Nucleotide excision repair (NER) is responsible for removing a wide variety of bulky adducts from DNA, thus contributing to the maintenance of genome stability. The efficiency with which proteins of the NER system recognize and remove bulky adducts depends on many factors and is of great clinical and diagnostic significance. The review examines current concepts of the NER system molecular basis in eukaryotic cells and analyzes methods for the assessment of the NER-mediated DNA repair efficiency both in vitro and ex vivo.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.

Bulky Adducts in Clustered DNA Lesions: Causes of Resistance to the NER System.

The nucleotide excision repair (NER) system removes a wide range of bulky DNA lesions that cause significant distortions of the regular double helix structure. These lesions, mainly bulky covalent DNA adducts, are induced by ultraviolet and ionizing radiation or the interaction between exogenous/endogenous chemically active substances and nitrogenous DNA bases. As the number of DNA lesions increases, e.g., due to intensive chemotherapy and combination therapy of various diseases or DNA repair impairment, clustered lesions containing bulky adducts may occur. Clustered lesions are two or more lesions located within one or two turns of the DNA helix. Despite the fact that repair of single DNA lesions by the NER system in eukaryotic cells has been studied quite thoroughly, the repair mechanism of these lesions in clusters remains obscure. Identification of the structural features of the DNA regions containing irreparable clustered lesions is of considerable interest, in particular due to a relationship between the efficiency of some antitumor drugs and the activity of cellular repair systems. In this review, we analyzed data on the induction of clustered lesions containing bulky adducts, the potential biological significance of these lesions, and methods for quantification of DNA lesions and considered the causes for the inhibition of NER-catalyzed excision of clustered bulky lesions.

Quantification of Urinary Thymidine Dimers in Volunteers After Ultraviolet Radiation Using a New UPLC-MS/MS-based Method.

Solar ultraviolet radiation (UVR) is a carcinogen and irradiation of the skin results in DNA damage. Cyclobutane pyrimidine dimers (CPDs), including thymidine dimers, are among the most frequent forms of DNA damage. When CPDs are formed, the nucleotide excision repair system is activated and CPDs are excreted in the urine. Here, we developed a mass spectrometry-based method to quantify thymidine dimers in the urine and tested the method on a small group of volunteers after whole-body UVR exposure. Years of research resulted in a method based on the "dilute-and-shoot" principle and ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. The whole body of each of eight healthy volunteers was exposed to 1.5-2.0 standard erythema doses (SEDs) of UVR for 3 consecutive days. Morning urine was collected on Day 1 (before irradiation) and on the following 7-9 days. Prior to analysis, sample preparation consisted of a simple dilution. A tandem quadrupole mass spectrometer coupled to UPLC was used for quantitative analysis in the multiple reaction monitoring mode. After 3 consecutive days of 1.5-2 SEDs, the highest level of thymidine dimer excretion occurred on Day 6 (0.7 ng/ml urine). Compared with baseline, significantly more thymidine dimers were excreted every day until Day 8 (p<0.016). Our method quantifies thymidine dimers that are excreted as dimers (i.e., not degraded further) after nucleotide excision repair. This is the first published mass spectrometry-based method for quantifying thymidine dimers in the urine after whole-body UVR exposure.

Chromatin-remodeling factor BAZ1A/ACF1 targets UV damage sites in an MLL1-dependent manner to facilitate nucleotide excision repair

Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the high-order DNA structure and prevent transcription and replication. The nucleotide excision repair (NER) system contributes to resolving this type of DNA lesion. There are two pathways that recognize pyrimidine dimers. One acts on transcribed strands of DNA (transcription-coupled NER), and the other acts on the whole genome (global genome-NER; GG-NER). In the latter case, DNA damage-binding protein 2 (DDB2) senses pyrimidine dimers with several histone modification enzymes. We previously reported that histone acetyltransferase binding to ORC1 (HBO1) interacts with DDB2 and facilitates recruitment of the imitation switch chromatin remodeler at UV-irradiated sites via an unknown methyltransferase. Here, we found that the phosphorylated histone methyltransferase mixed lineage leukemia 1 (MLL1) was maintained at UV-irradiated sites in an HBO1-dependent manner. Furthermore, MLL1 catalyzed histone H3K4 methylation and recruited the chromatin remodeler bromodomain adjacent to zinc finger domain 1A (BAZ1A)/ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1). Depletion of MLL1 suppressed BAZ1A accumulation at UV-irradiated sites and inhibited the removal of CPDs. These data indicate that the DDB2-HBO1-MLL1 axis is essential for the recruitment of BAZ1A to facilitate GG-NER.

Alteration of the Nucleotide Excision Repair (NER) Pathway in Soft Tissue Sarcoma.

Clinical responses to anticancer therapies in advanced soft tissue sarcoma (STS) are unluckily restricted to a small subgroup of patients. Much of the inter-individual variability in treatment efficacy is as result of polymorphisms in genes encoding proteins involved in drug pharmacokinetics and pharmacodynamics. The nucleotide excision repair (NER) system is the main defense mechanism for repairing DNA damage caused by carcinogens and chemotherapy drugs. Single nucleotide polymorphisms (SNPs) of NER pathway key genes, altering mRNA expression or protein activity, can be significantly associated with response to chemotherapy, toxicities, tumor relapse or risk of developing cancer. In the present study, in a cohort of STS patients, we performed DNA extraction and genotyping by SNP assay, RNA extraction and quantitative real-time reverse transcription PCR (qPCR), a molecular dynamics simulation in order to characterize the NER pathway in STS. We observed a severe deregulation of the NER pathway and we describe for the first time the effect of SNP rs1047768 in the ERCC5 structure, suggesting a role in modulating single-stranded DNA (ssDNA) binding. Our results evidenced, for the first time, the correlation between a specific genotype profile of ERCC genes and proficiency of the NER pathway in STS.

Genetic Variations in Nucleotide Excision Repair Pathway Genes and Risk of Allergic Rhinitis

Background Allergic rhinitis (AR) is the most frequent inflammatory disorder in the nasal mucosa that remains unclear etiology. Mounting studies suggested that genetic instability could trigger and worsen the inflammatory response. The nucleotide excision repair (NER) system is an important pathway in maintaining the stability of the genome. Therefore, the genetic variations in NER pathway genes may have potential effects on AR risk. Methods We evaluated the correlation between 19 candidate single nucleotide polymorphisms (SNPs) in NER pathway genes and AR susceptibility by a case-control study in a Chinese population, which contains 508 AR cases and 526 controls. Results Three independent SNPs were identified as significantly associated with AR susceptibility, including ERCC1 rs2298881 C > A (recessive model: adjusted odds ratios (OR) = 0.30, 95%confidence interval (CI) = 0.18–0.50, P < 0.0001), ERCC1 rs11615 G > A (dominant model: adjusted OR = 1.44, 95%CI = 1.04–2.01, P = 0.030), and XPC rs2228001 A > C (dominant model: adjusted OR = 0.68, 95%CI = 0.49–0.95, P = 0.024). Stratified analysis showed that ERCC1 rs2298881 AA genotype was correlated with a lower risk of AR among all the subgroups compared with rs2298881 CC/CA genotype. XPC rs2228001 AC/CC genotype reduced AR risk among the following subgroups: age > 60 months, clinical stage I and III. Conclusion Our finding showed that genetic variations in NER pathway genes: ERCC1 and XPC may affect the risk of AR, which will provide new insights into the genetics of AR from the perspective of DNA damage repair.

The UvrA-like protein Ecm16 requires ATPase activity to render resistance against echinomycin.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic‐producing bacteria. In this work, we characterize the self‐resistance protein Ecm16 encoded by echinomycin‐producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double‐stranded and single‐stranded) using a nucleotide‐independent binding mode. Ecm16’s binding affinity for DNA increased by 1.7‐fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16’s ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.

Antioxidant enzymes immobilized on gold and silver nanoparticles enhance DNA repairing systems of rat skin after exposure to ultraviolet radiation

The aim of the study was to investigate in vivo whether the application of immobilized superoxide dismutase (SOD) and catalase (CAT) could enhance DNA repairing systems and reduce level of CPD (cyclobutane pyrimidine dimers) and 6-4PP ((6-4) pyrimidine-pyrimidone photoproducts), and whether the immobilization on gold (AuNPs) and silver (AgNPs) nanoparticles affects the outcome. The study presents secondary analysis of our previous research. Three-day application of SOD and CAT in all forms of solution decreased the levels of CPD and 6-4PP boosted by UV irradiation. The mRNA expression level of the nucleotide excision repair (NER) system genes (XPA, XPC, ERCC1, ERCC2, ERCC3, LIG1) increased after application of immobilized and free enzymes. Increased by UV irradiation, p53 mRNA expression level normalized with the enzyme application. In conclusion, application of free and immobilized antioxidant enzymes accelerates removal of harmful effects of UV radiation in the rat skin by increasing expression level of NER genes.

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

UV Radiation in DNA Damage and Repair Involving DNA-Photolyases and Cryptochromes.

Prolonged exposure to ultraviolet radiation on human skin can lead to mutations in DNA, photoaging, suppression of the immune system, and other damage up to skin cancer (melanoma, basal cell, and squamous cell carcinoma). We reviewed the state of knowledge of the damaging action of UVB and UVA on DNA, and also the mechanisms of DNA repair with the participation of the DNA-photolyase enzyme or of the nucleotide excision repair (NER) system. In the course of evolution, most mammals lost the possibility of DNA photoreparation due to the disappearance of DNA photolyase genes, but they retained closely related cryptochromes that regulate the transcription of the NER system enzymes. We analyze the published relationships between DNA photolyases/cryptochromes and carcinogenesis, as well as their possible role in the prevention and treatment of diseases caused by UV radiation.

300 The time-dependency of the cyclobutane pyrimidine dimer-evoked cellular damages using a CPD-specific photolyase-encoding mRNA-based model system

UVB damages DNA predominantly by the formation of cyclobutane pyrimidine dimers (CPDs) that are repaired by nucleotide excision repair system in humans. Organisms more primitive than placental mammals remove CPDs by photolyase in a process of photoreactivation that uses the energy of visible light. Our previously established model system based on transient transfection of human keratinocytes with in vitro transcribed CPD-photolyase mRNA containing 1-methylpseudouridine modifications (CPD-PL mRNA).

Recognition and removal of clustered DNA lesions via nucleotide excision repair

Clustered damage of DNA consists of two or more lesions located within one or two turns of the DNA helix. Clusters consisting of lesions of various structures can arise under the influence of strong damaging factors, especially if the cells have a compromised repair status. In this work, we analyzed how the presence of an analog of the apurinic/apyrimidinic site – a non-nucleoside residue consisting of diethylene glycol phosphodiester (DEG) – affects the recognition and removal of a bulky lesion (a non-nucleoside site of the modified DNA strand containing a fluorescein residue, nFlu) from DNA by a mammalian nucleotide excision repair system. Here we demonstrated that the efficiency of nFlu removal decreases in the presence of DEG in the complementary strand and is completely suppressed when the DEG is located opposite the nFlu. By contrast, protein factor XPC-RAD23B, which initiates global genomic nucleotide excision repair, has higher affinity for DNA containing clustered damage as compared to DNA containing a single bulky lesion; the affinity of XPC strengthens as the positions of DEG and nFlu become closer. The changes in the double-stranded DNA’s geometry caused by the presence of clustered damage were also assessed. The obtained experimental data together with the results of molecular dynamics simulations make it possible to get insight into the structural features of DNA containing clustered lesions that determine the efficiency of repair. Speaking more broadly, this study should help to understand the probable fate of bulky adduct-containing clusters of various topologies in the mammalian cell.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

Toxicological Comparison of Mancozeb and Zoxamide Fungicides at Environmentally Relevant Concentrations by an In Vitro Approach.

Mancozeb (MZ) and zoxamide (ZOX) are fungicides commonly used in pest control programs to protect vineyards. Their toxic and genotoxic potential were investigated in vitro on HepG2 and A549 cell lines at environmentally relevant concentrations. Cytotoxicity, apoptosis, necrosis and intracellular reactive oxygen species (ROS), comet assay and a micronucleus test with CREST immunofluorescence were used. The expression of a panel of genes involved in apoptosis/necrosis (BAX/BCL2), oxidative stress (NRF2), drug metabolism (CYP1A1) and DNA repair (ERCC1/OGG1) was evaluated by real-time PCR. Both fungicides were cytotoxic at the highest tested concentrations (295.7 and 463.4 µM, respectively); MZ induced necrosis, ZOX did not increase apoptosis but modulated BAX and BCL2 expression, suggesting a different mechanism. Both compounds did not increase ROS, but the induction of CYP1A1 and NRF2 expression supported a pro-oxidant mechanism. The comet assay evidenced MZ genotoxicity, whereas no DNA damage due to ZOX treatment was observed. Positive micronuclei were increased in both cell lines treated with MZ and ZOX, supporting their aneugenic potential. ERCC1 and OGG1 were differently modulated, indicating the efficient activation of the nucleotide excision repair system by both fungicides and the inhibition of the base excision repair system by MZ. Overall, MZ confirmed its toxicity and new ZOX-relevant effects were highlighted.

The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage.

The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.

Current Therapeutic Strategies of Xeroderma Pigmentosum.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disease caused by a defect in the DNA repair system, exhibiting skin cancer on sun exposure. As it is an incurable disease, therapeutic strategies of this disease are critical. This review article takes an attempt to explore the current therapeutic advancements in XP. Different approaches including sun avoidance; surgical removal of cancerous lesions; laser and photodynamic therapy; use of retinoid, 5-fluorouracil, imiquimod, photolyase, and antioxidant; interferon therapy and gene therapy are chosen by doctors and patients to lessen the adverse effects of this disease. Among these options, sun avoidance, use of 5-fluorouracil and imiquimod, and interferon therapy are effective. However, some approaches including laser and photodynamic therapy, and the use of retinoids are effective against skin cancer having severe side effects. Furthermore, surgical removal of cancerous lesions and use of antioxidants are considered to be effective against this disease; however, efficacies of these are not experimentally determined. In addition, some approaches including oral vismodegib, immunotherapy, nicotinamide, acetohexamide, glimepiride-restricted diet are found to be effective to minimize the complications secondary to defects in the nucleotide excision repair (NER) system and also enhance the NER, which are under experimental level yet. Besides these, gene therapy, including the introduction of missing genes and genome edition, may be a promising approach to combat this disease, which is also not well established now. In the near future, these approaches may be effective tools to manage XP.