DNA Nucleotide Excision Repair Research Articles

Development of effective skin cancer treatment and prevention in xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a rare, recessively transmitted genetic disease characterized by increasingly marked dyspigmentation and xerosis (dryness) of sun-exposed tissues, especially skin. Skin cancers characteristically develop in sun-exposed sites at very much earlier ages than in the general population; these are often multiple and hundreds or even thousands may develop. Eight complementation groups have been identified. Seven groups, XP-A…G, are associated with defective genes encoding proteins involved in the nucleotide excision DNA repair (NER) pathway that recognizes and excises mutagenic changes induced in DNA by sunlight; the eighth group, XP-V, is associated with defective translesion synthesis (TLS) bypassing such alterations. The dyspigmentation, xerosis and eventually carcinogenesis in XP patients appear to be due to their cells' failure to respond properly to these mutagenic DNA alterations, leading to mutations in skin cells. A subset of cases, especially those in some complementation groups, may develop neurological degeneration, which may be severe. However, in most XP patients, in the past the multiple skin cancers have led to death at an early age due to either metastases or sepsis. Using either topical 5-fluorouracil or imiquimod, we have developed a protocol that effectively prevents most skin cancer development in XP patients.

Impact of age-associated cyclopurine lesions on DNA repair helicases.

8,5′ cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.

Abstract 3381: Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells

Abstract Background: ERCC4 and ERCC5 are key nucleotide excision DNA repair genes and are expressed abundantly in normal bronchial epithelial cells (NBEC). DNA sequence variation in ERCC4 or ERCC5 is associated with risk for lung cancer in multiple independent studies. C/EBPG expression is correlated with that of ERCC4, ERCC5 and many other key genes in BEC, suggesting a regulatory role, supported by experimental observation that up-regulation of CCAAT/enhancer-binding protein gamma (C/EBPG) transcription factor up-regulates ERCC5 expression in H23 lung cancer cell line. The purpose of this study was to test the hypothesis that knockdown of C/EBPG in lung cancer cells would reduce transcription of ERCC5 and ERCC4. Methods: We knocked-down C/EBPG transcript level by C/EBPG siRNA transfection in three non-small cell lung cancer cell lines: H23, H520 and H1703. Following transfection, RNA was extracted after 24 and 48 hours. Reduction of C/EBPG transcript abundance measured by competitive multiplex RT-PCR in H23, H520, and H1703 was 56%, 84%, and 92% at 24 hours, and 82%, 89%, and 76% at 48 hours. After confirming C/EBPG knockdown, we measured allele-specific expression (ASE) and total expression of C/EBPG, ERCC4 ERCC5 through competitive multiplex PCR-based amplicon sequencing library preparation followed by Illumina HiSeq next generation sequencing (NGS) (Blomquist et al, PLOS one, 2013). This NGS method a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. ASE was measured at ERCC4 SNPs rs2276466, rs3743538 and ERCC5 SNPs rs1047768, rs17655, rs4150316. Results: In H23 C/EBPG knock-down was associated with no change in expression at any of the SNPs for ERCC4 or ERCC5, while in H520 one ERCC5 rs1047768 allele decreased (3-fold) and the other allele had no change, and in H1703 both ERCC5 rs1047768 alleles increased (10-fold, and 2-fold). In H520, ERCC4 measured at two SNPs were different with increase at one allele but not the other at rs3743538 and decrease for both alleles at rs2276466. In H1703 each allele at ERCC4 rs3743538 increased, 2-fold and 45-fold respectively and each allele at ERCC4 rs2276466 increased, 5-fold and 2.7-fold respectively. Conclusions: These results support prior evidence that C/EBPG regulates ERCC5 transcription, and provides evidence that it also contributes to regulation of ERCC4. The inter-allelic and inter-cell line variation in response to C/EBPG knockdown data supports that hypothesis that cis-regulatory DNA variants interact with C/EBPG in regulation of these genes. The lack of response in H23 may be in part due to the low constitutive level of ERCC4 and ERCC5 expression in this cell line. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3381. doi:10.1158/1538-7445.AM2014-3381

Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding.

Rad2/XPG belongs to the flap nuclease family and is responsible for a key step of the eukaryotic nucleotide excision DNA repair (NER) pathway. To elucidate the mechanism of DNA binding by Rad2/XPG, we solved crystal structures of the catalytic core of Rad2 in complex with a substrate. Rad2 utilizes three structural modules for recognition of the double-stranded portion of DNA substrate, particularly a Rad2-specific α-helix for binding the cleaved strand. The protein does not specifically recognize the single-stranded portion of the nucleic acid. Our data suggest that in contrast to related enzymes (FEN1 and EXO1), the Rad2 active site may be more accessible, which would create an exit route for substrates without a free 5′ end.

Association of xeroderma pigmentosum complementation group G Asp1104His polymorphism with breast cancer risk: A cumulative meta-analysis.

The xeroderma pigmentosum complementation group G (XPG) gene plays an important role in the DNA nucleotide excision repair (NER) pathway. Several studies have investigated the association between the XPG Asp1104His polymorphism and breast cancer; however, the results have been inconsistent. Therefore, we conducted a meta-analysis of 8 published articles (10 case-control studies) including a total of 5,235 patients with breast cancer and 5,685 healthy controls. The results demonstrated that the XPG Asp1104His polymorphism was not associated with breast cancer in the overall population [His vs. Asp, odds ratio (OR)=1.00, 95% confidence interval (CI): 0.91-1.08; His/His vs. Asp/Asp, OR=0.96, 95% CI: 0.83-1.11; Asp/His vs. Asp/Asp, OR=1.02, 95% CI: 0.94-1.11; His/His+Asp/His vs. Asp/Asp, OR=1.03, 95% CI: 0.92-1.15; and His/His vs. Asp/Asp+Asp/His, OR=0.93, 95% CI: 0.81-1.06]. In the subgroup analysis by ethnicity, no significant association was observed in European subjects. In conclusion, this meta-analysis suggested that the XPG Asp1104His polymorphism is not associated with breast cancer risk.

Polymorphism of DNA repair genes OGG1, XRCC1, XPD and ERCC6 in bladder cancer in Belarus

Context: The study of DNA base and nucleotide excision repair gene polymorphisms in bladder cancer seems to have a predictive value because of the evident relationship between the DNA damage response induced by environmental mutagens and cancer predisposition.Objective: The objective was to determine OGG1 Ser326Cys, XRCC1 Arg399Gln, XPD Asp312Asn, and ERCC6 Met1097Val polymorphisms in bladder cancer patients as compared to controls.Methods: Both groups were predominantly represented by Belarusians and Eastern Slavs. DNA samples from 336 patients and 370 controls were genotyped using a PCR-RFLP method.Results: The genotype distributions were in agreement with the Hardy–Weinberg equilibrium. The minor allele frequencies in the control population were in the range of those in Caucasians in contrast to Asians. The OGG1 326 Ser/Cys and XPD 312 Asp/Asn heterozygous genotypes were inversely associated with cancer risk (OR [95% CI] = 0.69 [0.50–0.95] and 1.35 [1.0–1.82], respectively). The contrasting effects of these genotypes were potentiated due to their interactions with smoking habit or age.Conclusions: Among four DNA repair gene polymorphisms, the OGG1 326 Ser/Cys and XPD 312 Asp/Asn heterozygous genotypes might be recognized as potential genetic markers modifying susceptibility to bladder cancer in Belarus.

Fluorescence detection of cellular nucleotide excision repair of damaged DNA.

To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6–4) photoproduct and a fluorophore–quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6–4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.

Prognostic/predictive biomarkers in advanced soft tissue sarcomas (STS): Translational research associated to randomized phase II trial comparing trabectedin-doxorubicin versus doxorubicin—A GEIS study.

10500 Background: Nucleotide excision and homologous recombination DNA repair pathways are the most recognized antitumor mechanism of action of trabectedin in STS. Additionally, our group has shown...

Defective Mitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1 Reduction

Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA-deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a nuclear-mitochondrial crosstalk that is critical for the maintenance of mitochondrial health.

XPF-673C>T Polymorphism Effect on the Susceptibility to Esophageal Cancer in Chinese Population

PurposeXeroderma pigmentsum group F (XPF) plays a pivotal role in DNA nucleotide excision repair and has been linked to the development of various cancers. This study aims to assess the association of XPF genetic variants with the susceptibility to esophageal squamous cell carcinoma (ESCC) in Chinese population.MethodsThis two-stage case-control study was conducted in a total of 1524 patients with ESCC and 1524 controls. Genotype of XPF -673C>T and 11985A>G variants were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis was performed to estimate odd ratios (ORs) and 95% confidence intervals (95% CI).ResultsOur case-control study showed that XPF -673TT genotype was associated with a decreased risk of ESCC compared with CC genotype in both case-control sets (Tangshan set: OR = 0.58; 95%CI = 0.34–0.99, P = 0.040; Beijing set: OR = 0.66; 95%CI = 0.46–0.95, P = 0.027). Stratified analyses revealed that a multiplicative interaction between -673C>T variant and age, sex or smoking status was evident (Gene-age: Pinteraction = 0.002; Gene-sex: Pinteraction = 0.002; Gene-smoking: Pinteraction = 0.002). For XPF 11985A>G polymorphism, there was no significant difference of genotype distribution between ESCC cases and controls.ConclusionThese findings indicated that genetic variants in XPF might contribute to the susceptibility to ESCC.

Characterization and differential expression of CPD and 6–4 DNA photolyases in Xiphophorus species and interspecies hybrids

Among the many Xiphophorus interspecies hybrid tumor models are those that exhibit ultraviolet light (UVB) induced melanoma. In previous studies, assessment of UVB induced DNA damage and nucleotide excision DNA repair has been performed in parental lines and interspecies hybrids. Species and hybrid specific differences in the levels of DNA damage induced and the dark repair rates for cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine pyrimidine photoproducts (6–4PPs) have been reported. However, UVB induced DNA lesions in Xiphophorus fishes are thought to primarily be repaired via light dependent CPD and 6–4PP specific photolyases. Photolyases are of evolutionary interest since they are ancient and presumably function solely to ameliorate the deleterious effects of UVB exposure. Herein, we report results from detailed studies of CPD and 6-4PP photolyase gene expression within several Xiphophorus tissues. We determined photolyase gene expression patterns before and after exposure to fluorescent light in X. maculatus, X. couchianus, and for F1 interspecies hybrids produced from crossing these two parental lines (X. maculatus Jp 163 B×X. couchianus). We present novel results showing these two photolyase genes exhibit species, tissue, and hybrid-specific differences in basal and light induced gene expression.

Circulation Editors’ Picks

<i>Circulation</i> Editors’ Picks

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

DNA crosslinks, DNA damage and repair in peripheral blood lymphocytes of non-small cell lung cancer patients treated with platinum derivatives

Lung cancer is the leading cause of cancer-related mortality in the world. Chemotherapy has been the mainstay of treatment for advanced non-small cell lung cancer (NSCLC) and platinum-based derivatives have been shown to improve overall survival. The aim of the present study was to investigate the DNA damage [single strand breaks (SSBs) and DNA crosslinks] and DNA repair in peripheral blood lymphocytes in patients with NSCLC treated with platinum derivatives using modified comet assay. Twenty patients in the final (4th) stage of NSCLC and 10 age-corresponding healthy controls participated in the study. Alkaline comet assay was performed according to the appropriate protocol. The DNA base excision repair (BER) activity of the controls was significantly higher compared to that of cancer patients, and the activity of DNA nucleotide excision repair (NER) was almost at the same level both in controls and patients. We observed changes in the amount of SSBs and DNA crosslinks during the course of chemotherapy. We found a significantly higher level of SSBs immediately after administration of chemotherapy. Similarly, we found the highest incidence of DNA crosslinks immediately or 1 day after chemotherapy (compared to measurement before chemotherapy). Moreover, we compared the levels of DNA repair in patients who survived chemotherapy with those in patients who died in the course of chemotherapy: the activity of BER was higher in the case of surviving patients, while the levels of NER were essentially the same. The data arising from the present study confirm the findings of other studies dealing with DNA damage and repair in cancer patients treated with chemotherapy. Moreover, our results indicated that despite the fact that cisplatin-DNA adducts are removed by the NER pathway, BER may also play a role in the clinical status of patients and their survival.

Erythropoietic Defect Associated with Reduced Cell Proliferation in Mice Lacking the 26S Proteasome Shuttling Factor Rad23b

Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the redundant function of the Rad23b paralogue, Rad23a. However, Rad23b-null midgestation embryos are anemic, and most embryos die before birth. Using an unbiased proteomics approach, we found that the majority of Rad23b-interacting partners are associated with the ubiquitin-proteasome system (UPS). We tested the requirement for Rad23b-dependent UPS activity in cellular proliferation and more specifically in the process of erythropoiesis. In cultured fibroblasts derived from embryos lacking Rad23b, proliferation rates were reduced. In fetal livers of Rad23b-null embryos, we observed reduced proliferation, accumulation of early erythroid progenitors, and a block during erythroid maturation. In primary wild-type (WT) erythroid cells, knockdown of Rad23b or chemical inhibition of the proteasome reduced survival and differentiation capability. Finally, the defects linked to Rad23b loss specifically affected fetal definitive erythropoiesis and stress erythropoiesis in adult mice. Together, these data indicate a previously unappreciated requirement for Rad23b and the UPS in regulation of proliferation in different cell types.

Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview

There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results. Here we provide a comprehensive summary of protocols for the comet-based BER- and NER-specific in vitro DNA repair assays that can be applied to a wide spectrum of biological material--cultured cell lines, blood cells, animal tissue samples and human biopsies. Our intention is to provide a detailed and user-friendly account of the assays, including practical tips and recommendations to help in setting them up. By proposing standard protocols, we hope to facilitate comparison of results obtained in different laboratories.

Repressing DNA Repair to Enhance Chemotherapy: Targeting MyD88 in Colon Cancer

Repressing DNA Repair to Enhance Chemotherapy: Targeting MyD88 in Colon Cancer

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D)

BackgroundTo investigate the association of DNA nucleotide excision repair (NER) defects with neurological degeneration, cachexia and cancer, we performed autopsies on 4 adult xeroderma pigmentosum (XP) patients with different clinical features and defects in NER complementation groups XP-A, XP-C or XP-D.ResultsThe XP-A (XP12BE) and XP-D (XP18BE) patients exhibited progressive neurological deterioration with sensorineural hearing loss. The clinical spectrum encompassed severe cachexia in the XP-A (XP12BE) patient, numerous skin cancers in the XP-A and two XP-C (XP24BE and XP1BE) patients and only few skin cancers in the XP-D patient. Two XP-C patients developed internal neoplasms including glioblastoma in XP24BE and uterine adenocarcinoma in XP1BE. At autopsy, the brains of the 44 yr XP-A and the 45 yr XP-D patients were profoundly atrophic and characterized microscopically by diffuse neuronal loss, myelin pallor and gliosis. Unlike the XP-A patient, the XP-D patient had a thickened calvarium, and the brain showed vacuolization of the neuropil in the cerebrum, cerebellum and brainstem, and patchy Purkinje cell loss. Axonal neuropathy and chronic denervation atrophy of the skeletal muscles were observed in the XP-A patient, but not in the XP-D patient.ConclusionsThese clinical manifestations and autopsy findings indicate advanced involvement of the central and peripheral nervous system. Despite similar defects in DNA repair, different clinicopathological phenotypes are seen in the four cases, and therefore distinct patterns of neurodegeneration characterize XP-D, XP-A and XP-C patients.

Cockayne syndrome pathogenesis: Lessons from mouse models

Cockayne syndrome (CS) is a rare multisystem disorder characterized by cachectic dwarfism, nervous system abnormalities and features of premature aging. CS symptoms are associated with mutations in 5 genes, CSA, CSB, XPB, XPD and XPG encoding for proteins involved in the transcription-coupled subpathway of nucleotide excision DNA repair (NER). Mutant mice have been generated for all CS-associated genes and provide tools to examine how the cellular defects translate into CS symptoms. Mice deficient for Csa or Csb genetically mimic CS in man, and develop mild CS symptoms including reduced fat tissue, photoreceptor cell loss, and mild, but characteristic, nervous system pathology. These mild CS models are converted into severe CS models with short life span, progressive nervous system degeneration and cachectic dwarfism after simultaneous complete inactivation of global genome NER. A spectrum of mild-to-severe CS-like symptoms occurs in Xpb, Xpd, and Xpg mice that genetically mimic patients with a disorder that combines CS symptoms with another NER syndrome, xeroderma pigmentosum. In conclusion, CS mouse models mice develop a range of CS phenotypes and open promising perspectives for testing interventional approaches.