DNA Nucleotide Excision Repair Research Articles

Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (K(D) ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nM, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA–D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB–DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC–DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

Modulation of DNA base excision repair during neuronal differentiation

Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because they have robust BER capacity, which is heavily attenuated in postmitotic neurons. The reduction in BER activity in differentiated cells correlates with diminished protein levels of key long patch BER components, flap endonuclease-1, proliferating cell nuclear antigen, and ligase I. Thus, because of their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared with their neuronally differentiated progeny.

Trichothiodystrophy in a child with occult learning disorder

Trichothiodystrophy (TTD) is an autosomal recessive condition characterized by brittle and sparse sulfur deficient hair. The disorder is due to a known genetic mutation in DNA nucleotide excision repair (NER) in up to 83% of cases. We describe a 13-month-old girl presenting with hair fragility and hair loss since age 3 months, and discuss the overlap between TTD and other NER diseases. This case report highlights the importance of early diagnosis of occult learning disorder in young children with TTD and the need for early assessment and involvement of multidisciplinary team to target the child's educational needs.

Oxidative-stress-induced epigenetic changes in chronic diabetic complications

Oxidative stress plays an important role in the development and progression of chronic diabetic complications. Diabetes causes mitochondrial superoxide overproduction in the endothelial cells of both large and small vessels. This increased superoxide production causes the activation of several signal pathways involved in the pathogenesis of chronic complications. In particular, endothelial cells are major targets of glucose-induced oxidative damage in the target organs. Oxidative stress activates cellular signaling pathways and transcription factors in endothelial cells including protein kinase C (PKC), c-Jun-N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), forkhead box O (FOXO), and nuclear factor kappa-B (NF-κB). Oxidative stress also causes DNA damage and activates DNA nucleotide excision repair enzymes including the excision repair cross complimenting 1(ERCC1), ERCC4, and poly(ADP-ribose) polymerase (PARP). Augmented production of histone acetyltransferase p300, and alterations of histone deacetylases, including class III deacetylases sirtuins, are also involved in this process. Recent research has found that small noncoding RNAs, like microRNA, are a new kind of regulator associated with chronic diabetic complications. There are extensive and complicated interactions and among these molecules. The purpose of this review is to demonstrate the role of oxidative stress in the development of diabetic complications in relation to epigenetic changes such as acetylation and microRNA alterations.

Oxidative DNA Damage and Nucleotide Excision Repair

Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues.

Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

Escherichia coli pol V (UmuD′2C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by umuC_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV in vivo. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis.

Abstract IA10: Deciphering the signals from the GWAS hits: From function to epidemiology and back again.

Abstract Genome wide association studies (GWAS) have identified hundreds of single nucleotide polymorphisms (SNPs) that confer susceptibility to a multitude of complex, multifactorial disease traits including cardiovascular disease, diabetes, and several cancers. For the vast majority of identified common genetic variants, the functional rationale underlying disease is largely unknown. The functional effects of common risk associated SNPs are likely to be subtle given that they confer smaller risks (ORs <1.2). More than 90% of confirmed common risk-SNPs lie in non-coding DNA, often several kilobases from the nearest gene suggesting a critical role for non-coding DNA in the functional influence of these SNPs. A significant focus of functional follow-up studies at common susceptibility loci has therefore been to understand the interplay between non-coding DNA elements such as regulators and enhancers, non-coding RNA species such as micro- and long non-coding RNAs, and risk associated germline variants. GWAS studies have also highlighted regions of the genome that contain plausible cancer susceptibility genes that may be specific to disease etiology (e.g. the FGFR gene in breast cancer), and other genes that may play more general role in cancer (e.g. the TERT gene, multi-cancer risk locus). The current presentation will focus on ovarian cancer. The Ovarian Cancer Association Consortium (OCAC) has identified 11 confirmed susceptibility loci, and suggest multiple candidate genes that have either previously been implicated in ovarian cancer development, or function in pathways such as double strand DNA break and nucleotide excision repair, which suggests they may play a significant role in ovarian cancer risk and/or clinical outcome. Data presented will describe approaches to understanding the functional role of both susceptibility SNPs and candidate genes in the underlying cellular etiology of different subtypes of ovarian cancer, and provide evidence suggesting a role of candidate genes in disease initiation and treatment response. Overall, these studies highlight the challenges we face in the developing field of functional-epidemiology, in particular in understanding common low-penetrance susceptibility loci. However, the emerging data also provide support for the role of GWAS in unearthing critical, previously unknown mechanisms that underlie cancer biology, suggesting these studies may have a substantial clinical impact in the future. Citation Format: Simon A. Gayther. Deciphering the signals from the GWAS hits: From function to epidemiology and back again. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA10.

Effects of compound heterozygosity at the Xpd locus on cancer and ageing in mouse models

XPD is a helicase subunit of transcription factor IIH, an eleven-protein complex involved in a wide range of cellular activities including transcription and nucleotide excision DNA repair (NER). Mutations in NER genes including XPD can lead to a variety of overlapping syndromes with three general categories of symptoms in addition to sun (UV) sensitivity: severe skin cancer predisposition as in xeroderma pigmentosum (XP), segmental progeria as in trichothiodystrophy (TTD) and Cockayne syndrome (CS), and a combination of both as in XP/CS and XP/TTD. Genetic background and compound heterozygosity are two factors potentially complicating straightforward interpretations of genotype–phenotype relationship at the XPD locus. Previously we showed that the presence of two different mutant Xpd alleles in compound heterozygous mice could in principle contribute to disease heterogeneity through biallelic effects, including dominance of one mutant allele over another and interallelic complementation between mutant alleles, in a tissue-specific manner. Here we report on the interaction between different mutant alleles in compound heterozygous mice carrying one XP/CS-associated allele (XpdG602D) and one TTD-associated allele (XpdR722W) relative to homozygous controls in an isogenic background over a range of metabolic and UV-induced DNA damage-related phenotypes. We found complementation of metabolic phenotypes including body weight and insulin sensitivity, but none for any of the measured responses to UV irradiation. Instead, we found dominance of the partially functional TTD allele over the XPCS allele in most aspects of the response to UV irradiation including sunburn and skin cancer in vivo or cellular proliferation and DNA damage foci formation in vitro. These data support to a model of genotype–phenotype relationship at the XPD locus in which interactions between different recessive diseases alleles are a potent source of disease heterogeneity in compound heterozygous patients.

Low intensity infrared laser effects on Escherichia coli cultures and plasmid DNA

Biostimulative effect of low intensity laser in tissues has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases. The aim of this work was to evaluate effects of laser exposure on the survival of Escherichia coli cultures and plasmid topological forms. Escherichia coli cultures and plasmids were exposed to infrared laser to study bacterial survival and electrophoretic profile, respectively. Data indicate low intensity infrared laser: (i) had no effect on E. coli wild type, endonuclease IV, exonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cultures, but decreased the survival of E. coli UvrA protein deficient cultures; (ii) there was no alteration in the electrophoretic profile of plasmids. Exposure to low intensity infrared laser decreases survival of Escherichia coli cultures deficient in nucleotide excision repair of DNA and this effect could depend on fluences, wavelength and tissues conditions.

The association between polymorphisms in the DNA nucleotide excision repair genes and RRM1 gene and lung cancer risk.

Background Although numerous studies have investigated the association between DNA repair gene variants and lung cancer risk, the results remain inconclusive and incomplete. Methods We examined variants in seven nucleotide excision repair (NER) and RRM1 genes in association with primary lung cancer risk among 385 patients with newly diagnosed primary non-small cell lung cancer and 208 cancer-free controls collected from 2007 to 2009 in east China. The relationship between single nucleotide polymorphisms and risk of lung cancer was assessed by logistic regression and stratification analysis. Results The rs17655GG (ERCC5) and rs5744751CT (POLE) were associated with increased risk of lung cancer (adjusted odds ratio [OR], 2.32, 95% confidence interval [CI], 1.41-3.83; adjusted OR, 2.84, 95% CI, 1.70-4.74]. In stratification analysis, we found the increased effect of rs17655GG on the risk of lung cancer was observed among female, older subjects, or non-smokers. The heterozygote computed tomography (CT) of rs5744751 had a stronger unprotective effect on lung cancer among male, older subjects, or heavy smokers. Conclusion These results suggest that rs17655GG and rs5744751CT may be associated with lung cancer susceptibility in the Chinese population. However, these findings still need to be verified in larger confirmatory studies.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Nucleotide Excision DNA Repair Is Associated With Age-Related Vascular Dysfunction

Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1(d/-) and Xpd(TTD) mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1(d/-) mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1(d/-) mice, age-related endothelium-dependent vasodilator dysfunction in Xpd(TTD) animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells

Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.

TFIIH: when transcription met DNA repair

The transcription initiation factor TFIIH is a remarkable protein complex that has a fundamental role in the transcription of protein-coding genes as well as during the DNA nucleotide excision repair pathway. The detailed understanding of how TFIIH functions to coordinate these two processes is also providing an explanation for the phenotypes observed in patients who bear mutations in some of the TFIIH subunits. In this way, studies of TFIIH have revealed tight molecular connections between transcription and DNA repair and have helped to define the concept of 'transcription diseases'.

The Structure of the XPF-ssDNA Complex Underscores the Distinct Roles of the XPF and ERCC1 Helix- Hairpin-Helix Domains in ss/ds DNA Recognition

Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.

XPA A23G polymorphism and susceptibility to cancer: a meta-analysis

Xeroderma pigmentosum group A (XPA) participates in modulating recognition of DNA damage during the DNA nucleotide excision repair process. The XPA A23G polymorphism has been investigated in case-control studies to evaluate the cancer risk attributed to the variant, but the results were conflicting. To clarify the effect of XPA A23G polymorphism in cancer risk, we conducted a meta-analysis that included 30 published case-control studies. Overall, no significant association of XPA A23G variant with cancer susceptibility was observed for any genetic model. However, significant association was observed for colorectal cancer (GG vs. AA: OR = 1.68, 95% CI = 1.15-2.44; dominant genetic model GG + AG vs. AA: OR = 1.54, 95% CI = 1.08-1.17), for breast cancer an increased but non-significant risk was found (GG vs. AA: OR = 1.27, 95% CI = 0.98-1.66; dominant genetic model GG + AG vs. AA: OR = 1.27, 95% CI = 0.99-1.63), and for head and neck cancer an increased risk was observed in recessive model (OR = 1.19, 95% CI = 1.02-1.38), whereas for lung cancer a significant reduced risk was observed (GG vs. AA: OR = 0.77, 95% CI = 0.66–0.90; dominant genetic model GG + AG vs. AA: OR = 0.76, 95% CI = 0.66-0.87), it’s noting that in Asian population the inverse association was more apparent. In addition, in Asian population for esophageal cancer a significant decreased risk was also found in dominant genetic model (OR = 0.55; 95% CI = 0.43-0.70) and for head and neck cancer an increased risk was observed in dominant genetic model (OR = 1.51, 95% CI = 1.03-2.23). The meta-analysis suggested that the XPA A23G G allele is a low-penetrant risk factor for cancer development.

Damage recognition in nucleotide excision DNA repair

Nucleotide excision repair (NER) is a highly versatile DNA repair process. Its ability to repair a large number of different damages with the same subset of recognition factors requires structural tools for damage recognition that are both broad and very accurate. Over the past few years detailed structural information on damage recognition factors from eukaryotic and prokaryotic NER has emerged. These structures shed light on the toolkit utilized in the damage recognition process and help explain the broad substrate specificity of NER.

Epidermal Pigmentation, Nucleotide Excision Repair and Risk of Skin Cancer

As a group, malignancies of the skin are the most commonly diagnosed cancers, with over a million cases each year identified in the United States alone. While the keratinocyte malignancies – basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) – represent the majority of clinical cases of skin cancer, malignant melanoma is the most deadly form of skin cancer. Each of these skin malignancies is clearly linked to UV radiation, with chronic cumulative UV exposure being most relevant for keratinocyte malignancies and intense, blistering sunburns most relevant for melanoma. In this review, we describe the epidemiology of skin cancer, the link with UV radiation, and the innate defenses used to resist UV damage with particular attention to the nucleotide excision DNA repair pathway. We also focus on the role of skin pigmentation and the molecular events that control melanization of the skin, since these signaling pathways appear to be major determinants of skin cancer risk. We are particularly interested in the melanocortin 1 receptor (MC1R) signaling pathway which influences basal skin pigmentation, the ability to tan and the efficiency by which UV-induced photolesions are repaired in melanocytes after UV exposure.