DNA N-glycosylase Research Articles

Unraveling novel mRNA transcripts of the human DNA N-glycosylase 1 (NTHL1) gene with the implementation of an innovative targeted DNA-seq assay

The human NTHL1 gene encodes a DNA glycosylase that plays a key role in the base excision repair (BER) pathway, repairing oxidative DNA damage and maintaining genome integrity. The physiological activity of NTHL1 is crucial in preventing genetic alterations that can lead to cancer. In this study, we employed an innovative targeted DNA sequencing (DNA-seq) methodology to explore the transcriptional landscape of the NTHL1 gene, revealing previously uncharacterized alternative splicing events and novel exons. Our designed approach provided significantly improved sequencing depth and coverage, enabling the identification of novel NTHL1 mRNA transcripts. Bioinformatics analysis confirmed all annotated splice junctions of the main NTHL1 transcripts (v.1 – v.3) and revealed novel mRNA transcripts (NTHL1 v.4 – v.9) derived from splicing events between annotated exons as well as mRNAs containing previously uncharacterized exons (NTHL1 v.10 – v.14). Quantitative PCR analysis highlighted a diverse expression pattern of these novel transcripts across different human cell lines, suggesting cell-specific roles and regulatory mechanisms. Notably, NTHL1 v.5 was overexpressed in luminal A breast cancer cells (MCF-7), while v.13 was prominent in triple negative (BT-20), HER2 + breast cancer (SK-BR-3), prostate, colorectal cancer cells and HEK-293 cells. Our findings suggest that specific novel NTHL1 transcripts may encode protein isoforms with distinct structural features, as indicated by ribosome profiling datasets, while others containing premature termination codons could function as long non-coding RNAs. These insights enhance our understanding of NTHL1 regulatory role and its potential as a biomarker and therapeutic target in human malignancies. This study underscores the importance of exploring the transcriptional diversity of NTHL1 to fully elucidate its role in cancer pathobiology.

DNA N-glycosylases Ogg1 and EndoIII as components of base excision repair in Plasmodium falciparum organelles

The integrity of genomes of the two crucial organelles of the malaria parasite — an apicoplast and mitochondrion in each cell − must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1leader-GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway.

Dysregulation of base excision repair factors associated with low tumor immunogenicity in head and neck cancer: implication for immunotherapy.

Head and neck squamous carcinoma (HNSCC) is caused by different exogenous risk factors including smoking cigarettes, alcohol consumption, and HPV infection. Base excision repair (BER) is the frontline to repair oxidative DNA damage, which is initiated by the DNA N-glycosylase proteins (OGG1) and other BER factors including DNA polymerase β (POLB). Explore whether BER genes' (OGG1, POLB) overexpression in HNSCC alters genomic integrity, immunogenicity, and its role in prognostic value. RNA sequencing (RNA-Seq) and clinical information (age, gender, histological grade, survival status, and stage) of 530 patients of HNSCC were retrieved from the Cancer Genome Atlas. Patients' data are categorized HPV positive or negative to analyze the tumor data including the tumor stage, POLB, and OGG1 gene expression. RNA-Seq of HNSCC data retrieved and mutation count and aneuploidy score were compared using an unpaired t-test. The TIMER algorithm was used to calculate the tumor abundance of six infiltrating immune cells (CD4+ T cells, CD8+ T cells, B cells, neutrophils, macrophages, and dendritic cells) based on RNA-Seq expression profile data. The correlation between the POLB, OGG1, and immune cells was calculated by Spearman correlation analysis using TIMER 2.0. Our data analysis reveals that BER genes frequently overexpressed in HNSCC tumors and increase mutation count. In addition, OGG1 and POLB overexpression are associated with low infiltration of immune cells, low immune checkpoint gene expression (PD-1, cytotoxic T-lymphocyte antigen 4, program death ligand 1, and program death ligand 2), and innate immune signaling genes. Furthermore, dysregulated BER factors in Human papillomavirus (HPV) positive tumors had better overall survival. Our analysis suggests that dysregulation of the BER genes panel might be a potential prognosis marker and/or an attractive target for an immune checkpoint blockade in HNSCC cancers. However, our observation still requires further experimental-based scientific validation studies.

Functional Characterization of DNA N-Glycosylase Ogg1 and Ntg1 in DNA Damage Stress of Cryptococcus neoformans.

Reactive oxygen species induce DNA strand breaks and DNA oxidation. DNA oxidation leads to DNA mismatches, resulting in mutations in the genome if not properly repaired. Homologous recombination (HR) and non-homologous end-joining (NHEJ) are required for DNA strand breaks, whereas the base excision repair system mainly repairs oxidized DNAs, such as 8-oxoguanine and thymine glycol, by cleaving the glycosidic bond, inserting correct nucleotides, and sealing the gap. Our previous studies revealed that the Rad53-Bdr1 pathway mainly controls DNA strand breaks through the regulation of HR- and NHEJ-related genes. However, the functional roles of genes involved in the base excision repair system remain elusive in Cryptococcus neoformans. In the present study, we identified OGG1 and NTG1 genes in the base excision repair system of C. neoformans, which are involved in DNA oxidation repair. The expression of OGG1 was induced in a Hog1-dependent manner under oxidative stress. On the other hand, the expression of NTG1 was strongly induced by DNA damage stress in a Rad53-independent manner. We demonstrated that the deletion of NTG1, but not OGG1, resulted in elevated susceptibility to DNA damage agents and oxidative stress inducers. Notably, the ntg1Δ mutant showed growth defects upon antifungal drug treatment. Although deletion of OGG1 or NTG1 did not increase mutation rates, the mutation profile of each ogg1Δ and ntg1Δ mutant was different from that of the wild-type strain. Taken together, we found that DNA N-glycosylase Ntg1 is required for oxidative DNA damage stress and antifungal drug resistance in C. neoformans.

Microarray and in silico analysis of DNA repair genes between human testis of patients with nonobstructive azoospermia and normal cells.

DNA repair processes are critical to maintaining genomic integrity. As a result, dysregulation of repair genes is likely to be linked with health implications, such as an increased prevalence of infertility and an accelerated rate of aging. We evaluated all the DNA repair genes (322 genes) by microarray. This study has provided insight into the connection between DNA repair genes, including RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, INO80, PMS2L1, CHEK2, TRIP13, and POLD4. The microarray analysis of six human cases with different nonobstructive azoospermia revealed that RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, and INO80 were upregulated, and expression of PMS2L1, CHEK2, TRIP13, and POLD4 was downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluation was applied to predict proteins' functional and molecular interactions and then performed to recognize the master pathways. Functional enrichment analysis revealed that the biological process (BP) terms "base-excision repair, AP site formation,""nucleotide-excision repair, DNA gap filling," and "nucleotide-excision repair, preincision complex assembly" was significantly overexpressed in upregulated differentially expressed genes (DEGs). BP analysis of downregulated DEGs highlighted "histone phosphorylation,""DNA damage response, detection DNA response,""mitotic cell cycle checkpoint signaling,"and "double-strand break repair."Overrepresented molecular function (MF) terms in upregulated DEGs included "Oxidized base lesion DNA N-glycosylase activity,""uracil DNA N-glycosylase activity,""bubble DNA binding" and "DNA clamp loader activity."Interestingly, MF investigation of downregulated DEGs showed overexpression in "heterotrimeric G-protein complex,""5'-deoxyribose-5-phosphate lyase activity,""minor groove of adenine-thymine-rich DNA binding," and "histone kinase activity."Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Signal-on/signal-off bead-based assays for the multiplexed monitoring of base excision repair activities by flow cytometry.

As the support of all living kingdoms' genetic information, the integrity of the DNA biomolecule must be preserved. To that goal, cells have evolved specific DNA repair pathways to thwart a large diversity of chemical substances and radiations that alter the DNA structure and lead to the development of pathologies such as cancers or neurodegenerative diseases. When dysregulated, activity rates of various actors of DNA repair can play a key role in carcinogenesis as well as in drugs resistance or hypersensitivity mechanisms. For the last 10years, new complementary treatments have aimed at targeting specific enzymes responsible for such resistances. It is therefore crucial for biomedical research and clinical diagnosis to develop fast and sensitive tools able to measure the activity rate of DNA repair enzymes. In this work, a new assay for measuring enzymatic activities using microbeacons (µBs) is expounded. µB refers to microsphere functionalized by hairpin-shaped nucleic acid probes containing a single site-specific lesion in the stem and modified with chromophores. Following the processing of the lesion by the targeted protein, µB is cleaved and either lights off (signal-off strategy) or on (signal-on), depending on the use of fluorescent or profluorescent probes, respectively. After an optimization phase of the assay, we reported the combined analysis of restriction enzyme, AP-endonuclease, and DNA N-glycosylase by real-time monitoring followed by a flow cytometry measurement. As proofs of concept, we demonstrated the potential of the biosensor for highlighting DNA repair inhibitors and discriminating cell lines from their enzymatic activities.

Systematical Screening of Intracellular Protein Targets of Polyphemusin-I Using Escherichia coli Proteome Microarrays.

With their wide repertoire of mechanisms, antimicrobial peptides (AMPs) are promising alternatives to fight against varied pathogenic microorganisms (bacteria, fungi, viruses, parasites, etc.). AMPs, novel components of the innate immune defense system, are secreted by all organisms. The aquatic environment represents a huge population and an enormous source of varied AMPs. Polyphemusin-I, a marine AMP isolated from hemocytes of an American horseshoe crab, possesses high antimicrobial activities. Studies on polyphemusin-I have verified the intracellular mechanisms of action, however, its intracellular targets are not yet explored. In this study, we employed Escherichia coli proteome microarrays to systematically screen the entire intracellular protein targets of polyphemusin-I. A total of 97 protein targets of polyphemusin-I were statistically analyzed from the quadruplicate Escherichia coli proteome microarrays assays. Among these identified protein targets, 56 proteins had cellular location inside the cell (i.e., cytoplasm), one in the plasma membrane, one in the periplasm and the rest 39 proteins had no specified cellular location. The bioinformatics analysis of these identified protein targets of polyphemusin-I in gene ontology (GO) enrichment category of molecular function revealed significant enrichment in nucleic acid related GO terms i.e., “RNA binding”, “nucleotide binding”, “nuclease activities”, “uracil DNA N-glycosylase activities” and others. Moreover, enrichment in GO category of biological process also depicted enrichment in nucleic acid related GO terms, such as “nucleic acid phosphodiester bond hydrolysis”, “deoxyribonucleotide metabolism”, and others. In accordance to GO enrichment analysis, protein families (PFAM) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis also showed significant enrichment in nucleic acid terms. These enrichment results suggest that polyphemusin-I targets nucleic acid-associated proteins. Furthermore, to provide a comprehensive study, we compared the identified protein targets of polyphemusin-I with previously identified protein targets of four AMPs (P-Der, Lfcin B, PR-39, and Bac 7) using Escherichia coli proteome microarrays. The comparison study of five AMPs (polyhemusin-I, P-Der, Lfcin B, PR-39, and Bac 7) showed only nine common protein targets in all the five AMPs, whereas a total of 39 and 43 common protein targets were identified among the two marine AMPs (polyphemusin-I and P-Der) and three terrestrial AMPs (Lfcin B, PR-39 and Bac7), respectively. To further reveal the target pattern of marine and terrestrial AMPs, the enrichment results obtained from common protein targets of marine AMPs with terrestrial AMPs were compared. The comparison result indicated that AMPs have unique mechanism of action among marine or terrestrial AMPs. Hence, in this study, we have not only identified the intracellular protein targets of polyphemusin-I, but also revealed the protein target differences between marine AMPs and terrestrial AMPs.

Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil–DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth’s atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.

CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells.

CRISPR-guided DNA cytosine and adenine base editors (CBEs and ABEs) are widely used for many applications1–4 but primarily create DNA base transitions (i.e., pyrimidine-to-pyrimidine, or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an E. coli-derived uracil DNA N-glycosylase (eUNG), and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5, 6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

Overexpression of NEIL3 associated with altered genome and poor survival in selected types of human cancer.

Base excision repair, which is initiated by the DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. Several base excision repair genes are deregulated in cancer and affect cellular outcomes to chemotherapy and carcinogenesis. Endonuclease VIII-like 3 (NEIL3) is a DNA glycosylase protein that is involved in oxidative and interstrand crosslink DNA damage repair. Our previous work has showed that NEIL3 is required to maintain replication fork integrity. It is unknown whether NEIL3 overexpression could contribute to cancer phenotypes, and its prognostic value and use as potential drug target remain unexplored. Our analysis of cancer genomics data sets reveals that NEIL3 frequently undergoes overexpression in several cancers. Furthermore, patients who exhibited NEIL3 overexpression with pancreatic adenocarcinoma, lung adenocarcinoma, lower grade glioma, kidney renal clear cell carcinoma, and kidney papillary cell carcinoma had worse overall survival. Importantly, NEIL3 overexpressed tumors accumulate mutation and chromosomal variations. Furthermore, NEIL3 overexpressed tumors exhibit simultaneous overexpression of homologous recombination genes (BRCA1/2) and mismatch repair genes (MSH2/MSH6). However, NEIL3 overexpression is negatively correlated with tumor overexpressing nucleotide excision repair genes (XPA, XPC, ERCC1/2). Our results suggest that NEIL3 might be a potential prognosis marker for high-risk patients, and/or an attractive therapeutic target for selected cancers.

Significant Contribution of DNA Repair Human 8-Oxoguanine DNA N-Glycosylase 1 Genotypes to Renal Cell Carcinoma.

IntroductionDNA repair systems play essential roles in genomic stability and carcinogenesis. Therefore, genotypes at DNA repair loci may contribute to the determination of personal susceptibility to cancers. The contribution of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) genotypes to renal cell carcinoma (RCC) is largely unknown. This study aimed to evaluate the contributions of hOGG1 rs1052133 genotypes to the RCC risk.MethodsWe evaluated the contribution of hOGG1 rs1052133 (G/C) genotypes among 118 cases and 590 controls and analyzed the interactions of hOGG1 genotypes with smoking, alcohol drinking, hypertension, and diabetes status.ResultsThe hOGG1 rs1052133 CC genotype was significantly associated with a decreased RCC risk compared with that of the GG genotype (odds ratio [OR] = 0.25, 95% confidence interval [CI] = 0.09–0.72, p = 0.0049). The frequency of the rs1052133 C allele was significantly low in the RCC group (22.5% vs 31.2%; OR = 0.64; 95% CI = 0.46–0.89, p = 0.0074). Stratifying the analysis according to smoking, alcohol drinking, and diabetes status revealed no difference in the rs1052133 genotype distribution among these subgroups. A significant differential distribution of rs1052133 genotypes was observed among subjects with hypertension.ConclusionThe CC genotype of rs1052133 may play a role in determining RCC susceptibility among Taiwanese people and may serve as a biomarker of RCC, particularly in patients with hypertension.

Modification of the base excision repair enzyme MBD4 by the small ubiquitin-like molecule SUMO1

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

A benzimidazole-based ruthenium(IV) complex inhibits Pseudomonas aeruginosa biofilm formation by interacting with siderophores and the cell envelope, and inducing oxidative stress

Pseudomonas aeruginosa biofilm-associated infections are a serious medical problem, and new compounds and therapies acting through novel mechanisms are much needed. Herein, the authors report a ruthenium(IV) complex that reduces P. aeruginosa PAO1 biofilm formation by 84%, and alters biofilm morphology and the living-to-dead cell ratio at 1 mM concentration. Including the compound in the culture medium altered the pigments secreted by PAO1, and fluorescence spectra revealed a decrease in pyoverdine. Scanning electron microscopy showed that the ruthenium complex did not penetrate the bacterial cell wall, but accumulated on external cell structures. Fluorescence quenching experiments indicated strong binding of the ruthenium complex to both plasmid DNA and bovine serum albumin. Formamidopyrimidine DNA N-glycosylase (Fpg) protein digestion of plasmid DNA isolated after ruthenium(IV) complex treatment revealed the generation of oxidative stress, which was further proved by the observed upregulation of catalase and superoxide dismutase gene expression.

Role of endonuclease III enzymes in uracil repair

Endonuclease III is a DNA glycosylase previously known for its repair activity on oxidative pyrimidine damage. Uracil is a deamination product derived from cytosine. Uracil DNA N-glycosylase (UNG) and mismatch-specific uracil DNA glycosylase (MUG) are two known repair enzymes with enzymatic activity on uracil in E. coli. Here we report a G/U specific uracil DNA glycosylase activity in E. coli endonuclease III (endo III, Nth), which is comparable to MUG but significantly lower than its thymine glycol DNA glycosylase activity. The possibility that the novel activity is due to contamination is ruled out by expressing the wild type nth gene and an active site mutant in a uracil-repair-deficient genetic background. Consistent with the biochemical analysis, analyses of lac+ reversion and mutation frequencies in the presence of human AID induced cytosine deamination indicate the endo III can play a role in repair of cytosine deamination. In addition to E. coli, UDG activity is found in endo III homologs from other organisms. E. coli nucleoside diphosphate kinase (Ndk) was also tested for UDG activity because it was previously reported as an uracil repair enzyme. Under the assay conditions, very limited UDG activity was detected in single-stranded uracil-containing DNA from E. coli Ndk and no UDG activity was detected in human Ndk homologs. This study provides definitive clarification on uracil repair by endo III and reveals that endonuclease III is a G/U-specific UDG that can be viewed as a prototype for the human MBD4 uracil DNA glycosylase.

Evidence for Adaptive Response in a Molecular Epidemiological Study of the Inhabitants of a High Background-radiation Area of Yangjiang, China.

Our previous studies have shown that cancer mortality in high background-radiation areas of China was lower than that in a control area, indicating the possibility of an adaptive response in high background-radiation areas. Our aim is to determine the effect of low-dose radiation on the level of DNA oxidative damage, DNA damage repair, antioxidant capacity, and apoptosis in high background-radiation area and control area populations of Guangdong through a molecular epidemiological study in order to identify adaptive response. Blood samples were collected from male residents aged 50 to 59 y in a high background-radiation area (Yangjiang) and a control area (Enping), and activities of superoxide dismutase, glutathione, catalase, total antioxidant capacity, and expression of O6-methylguanine-DNA methyltransferase gene (MGMT), human 8-oxoguanine DNA N-glycosylase 1 gene (hOGG1), proapoptotic genes and antiapoptotic genes, oxidative-stress-related genes, as well as concentrations of 8-OHdG, TrxR, HSP27, and MT-COX2 were determined. The activities of antioxidative enzymes, relative mRNA expression level of DNA repair genes, antiapoptotic genes, oxidative-stress-related genes HSPB1 and MT-COX2, and the concentration of antioxidant index TrxR in the high background-radiation area population increased significantly compared to the control population (p < 0.05). The relative mRNA expression level of proapoptotic genes and the concentration of DNA oxidative damage index 8-OHdG were significantly lower in the high background-radiation area compared to those in the control area (p < 0.05). In conclusion, under long-term, natural, high background, ionizing radiation, DNA damage-repair capacity and antioxidant capacity of inhabitants in the high background-radiation area may be enhanced. Additionally, it could induce up regulation of cell-survival gene expression and down regulation of apoptotic gene expression. It might be speculated that enhanced antioxidant and DNA repair capacity and inhibition of apoptosis might play important roles in adaptive response of low-dose radiation in high background-radiation areas.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Polymorphism of 8-Oxoguanine DNA N-Glycosylase 1 Gene in Egyptian patients with Larynx Cancer

Polymorphism of 8-Oxoguanine DNA N-Glycosylase 1 Gene in Egyptian patients with Larynx Cancer

Thymine DNA Glycosylase (TDG) is involved in the pathogenesis of intestinal tumors with reduced APC expression.

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme that acts as a thymine and uracil DNA N-glycosylase on G:T and G:U mismatches, thus protecting CpG sites in the genome from mutagenesis by deamination. In addition, TDG has an epigenomic function by removing the novel cytosine derivatives 5-formylcytosine and 5-carboxylcytosine (5caC) generated by Ten-Eleven Translocation (TET) enzymes during active DNA demethylation. We and others previously reported that TDG is essential for mammalian development. However, its involvement in tumor formation is unknown. To study the role of TDG in tumorigenesis, we analyzed the effects of its inactivation in a well-characterized model of tumor predisposition, the ApcMin mouse strain. Mice bearing a conditional Tdgflox allele were crossed with Fabpl::Cre transgenic mice, in the context of the ApcMin mutation, in order to inactivate Tdg in the small intestinal and colonic epithelium. We observed an approximately 2-fold increase in the number of small intestinal adenomas in the test Tdg-mutant ApcMin mice in comparison to control genotypes (p=0.0001). This increase occurred in female mice, and is similar to the known increase in intestinal adenoma formation due to oophorectomy. In the human colorectal cancer (CRC) TCGA database, the subset of patients with TDG and APC expression in the lowest quartile exhibits an excess of female cases. We conclude that TDG inactivation plays a role in intestinal tumorigenesis initiated by mutation/underexpression of APC. Our results also indicate that TDG may be involved in sex-specific protection from CRC.

Graphistrength© C100 MultiWalled Carbon Nanotubes (MWCNT): thirteen-week inhalation toxicity study in rats with 13- and 52-week recovery periods combined with comet and micronucleus assays

Graphistrength© C100 provides superior electrical and mechanical properties for various applications and is one of the industrial MWCNT referenced in the OECD sponsorship program for the safety testing of nanomaterials. Graphistrength© C100 is formed of MWCNT (ca. 12 walls, outer mean diameter ca. 12 nm, length ca. 1 µm) agglomerated in particles with a granulometry centered on 400 µm. A general feature of MWCNT after inhalation or intratracheal exposures is the induction of an inflammatory reaction in the lungs sometimes associated with local genotoxic effects. Most of the in vitro and in vivo genotoxicity data available on Graphistrength© C100 are negative. However, a weak DNA damage activity in the in vitro and in vivo FPG-modified Comet assays and a weak clastogenic effect in the in vitro micronucleus test were reported. After investigating different parameters for the aerosol generation, male and female Wistar rats were exposed by nose-only inhalation (6h/day, 5d/week) to target concentrations of 0.05, 0.25 and 5.0 mg/m3 air of a respirable aerosol (MMAD < 3 µm) and sacrificed immediately after 4 and 13 weeks of exposure and 13 and 52 weeks of recovery after the 13-week exposure. Clinical, biological and histological evaluations were performed according to the OECD TG 413. Broncho-alveolar lavage fluid (BALF) was collected and analysed for cytokines and inflammatory parameters. Immediately after 13 weeks of exposure, chromosomal aberrations in the bone marrow cells of males and females were evaluated by the micronucleus test (OECD TG 474) and DNA damage in the lung, kidney and liver cells of males were assessed by both the standard and the human 8-oxoguanine DNA N-glycosylase 1 (hOGG1)-modified comet assay (OECD TG 489). Concentration-related deposition of black particles (MWCNT) was observed in lungs. At all sacrifice periods, an inflammatory lung reaction was observed in rats exposed to 5.0 mg/m3 associated with changes in the differential white blood cells counts. The lung inflammation was characterized by changes in the cytological, biochemical and cytokine parameters of the BALF, an increase of the lung weight, an interstitial inflammation mainly around the alveolar ducts at the bronchiole-alveolar junction and a cell hypertrophy/hyperplasia in the terminal and respiratory bronchioles. The slight changes in BALF parameters observed at 0.25 mg/m3 recovered after the 13-week treatment-free period and were not associated with any of the histological changes observed in lungs at 5.0 mg/m3. Signs of lung clearance of the MWCNT were observed at 0.05 and 0.25 mg/m3. After a one year treatment-free period, the inflammatory lung reaction was slight and of similar intensity that at the earlier sacrifice periods. Additional findings were minimal/slight bronchiolar/alveolar cell hypertrophy/hyperplasia and focally extensive alveolar septal fibrosis. No other pathological change was observed, nor was there any brain translocation via the olfactory bulb. The microscopic observations of the pleura were unremarkable. Neither increase in the number of micronucleated polychromatic erythrocytes nor increase in percent DNA damage were observed at any concentration. In conclusion, a lung inflammation characteristic of an overload with insoluble particles was observed after a 13-week inhalation exposure to 5.0 mg/m3 of Graphistrength© C100. A No-Observed Adverse Effect Concentration (NOAEC) of 0.25 mg/m3 was established for the repeated-dose toxicity and Graphistrength© C100 appears of low concern in term of local and systemic genotoxicity.