UNG Gene Research Articles

The association between inefficient repair of oxidative DNA lesions and common polymorphisms of the key base excision repair genes as well as their expression levels in patients with Rheumatoid Arthritis

IntroductionRA is a common autoimmune heterogeneous joint disease of still unknown etiology. One of the characteristic features of RA is oxidative stress, most probably induced and stimulated by inappropriate B and T-cell activity. We have hypothesized, that oxidative stress together with impaired DNA damage response to oxidative DNA lesions could be responsible for increased incidence in RA patients of some diseases with genetic instability background such as lymphoma.Material and methodsWe determined the level of oxidative DNA lesions and the kinetics of removal of DNA damage induced by TBH in PBMCs of 30 RA patients and 30 healthy individuals. The metrics from DNA repair study were correlated with genotypes of common polymorphisms of key BER genes as well as their expression levels. DNA repair were evaluated by comet assay, the genotypes of the polymorphism were determined by Taqman Assay and PrimeTime qPCR Assay was used to analyze the expression profile of genes related to BER.ResultsWe observed an association between RA occurrences and impaired DNA repair in PBMCs. After dividing the subjects by controls’ quartiles of DNA repair efficiency, we found an association between an increased risk of RA and inefficient DNA Repair. We also identified interaction between inefficient DNA Repair and polymorphism of the UNG gene (rs246079), and lower expression of key BER genes – MUTH, NEIL3 and UNG.ConclusionsOur result suggest that the genetic variations within BER genes as well as epigenetic factors may be linked with RA by the modulation of the cellular response to oxidative stress.

DNA Tamir Mekanizması İlişkili Genlerin Biyoinformatik Yöntemlerle Glioblastomda Tanımlanması

Amaç: DNA tamir mekanizmalarında (DTM) görev alan genlerin ifade değişimleri glioblastomda (GBM) radyoterapi direnci ile ilişkilendirilmiştir. DTM’de rol oynayan genlerin biyoinformatik yöntemlerle tanımlanması GBM tedavisinde kullanılabilecek potansiyel yeni hedeflerin belirlenmesine yardımcı olabilir. Bu çalışmanın amacı, DNA tamir mekanizmalarında rol oynayan genlerin biyoinformatik yöntemler kullanılarak GBM tümörlerinde tanımlanmasıdır. Yöntem: DNA tamiri mekanizmaları ile ilişkili genler “Reactome” ve “KEGG” veri tabanları üzerinde tanımlandı. GBM tümörlerinde genlere ait mRNA ifade profilleri GEO GDS1813 ve GDS2853 veri setlerinde “Orange Canvas” yazılımı kullanılarak incelendi. Genlerdeki genetik değişimler cBioPortal veri tabanı kullanılarak GBM TCGA olgularında tanımlandı. GEPIA2, değişen gen ifadelerinin TCGA GBM hasta sağ kalım süreleri üzerindeki etkisini göstermek için kullanıldı. Bulgular: ERCC6, FAN1, MBD4, PARP1 ve UNG genlerinin mRNA ifade profillerinin GBM tümörlerinde değişime uğradığı bulundu. Tanımlanan genler için farklı tipte mutasyonlar ve kopya sayı değişimleri TCGA GBM olgularında gözlendi. Yüksek ve düşük gen ifade profillerinin GBM hastalarının genel ve hastalıksız sağ kalım süreleri üzerinde etkisi olmadığı saptandı. Sonuç: Bu çalışmada tanımlanan ERCC6, PARP1 ve UNG genleri baskılanması durumunda GBM’de radyoterapi etkinliğini arttırabilecek potansiyel birer terapötik hedef olabilir.

Somatic Mutations in Oncogenes Are in Chronic Myeloid Leukemia Acquired De Novo via Deregulated Base-Excision Repair and Alternative Non-Homologous End Joining.

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.

A phage mechanism for selective nicking of dUMP-containing DNA

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

Evaluation of gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma.

This study aims to evaluate gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma both which have a distinct treatment and prognosis. Between January 2012 and January 2014, 12 newly diagnosed patients with a lung adenocarcinoma, 12 patients with malignant pleural mesothelioma, and eight healthy individuals as the control group were included. After treatment of the fresh samples of lung adenocarcinoma stored at -80°C for ribonucleic acid isolation, and paraffin-embedded tissues of patients with malignant pleural mesothelioma were deparaffinized, complementary deoxyribonucleic acid synthesis and expression of 84 genes associated with deoxyribonucleic acid repair were analyzed via real-time polymerase chain reaction assay. According to the expression of tumor cells, expression of each fold change was calculated. The BRCA1, BRCA2, CDK7, MLH3, MSH4, NEIL3, SMUG1, UNG, XRCC2, and XRCC4 genes showed more than five-fold higher expression in the patients with lung adenocarcinomas, compared to the control group. The patients with malignant pleural mesothelioma showed a five-fold higher expression in the APEX2, BRCA1, BRCA2, CDK7, MLH1, MLH3, MSH3, MSH4, NEIL3, PARP2, PARP3, PMS1, RAD50, RAD51, RAD51B, RAD51D, RAD52, RPA3, SMUG1, UNG, XPA, XRCC2, and XRCC4 genes, compared to the control group. Comparing malignant pleural mesothelioma with lung adenocarcinoma cases, we found that CDK7, MLH1, TREX1, PRKDC, XPA, PMS1, UNG, and RPA3 genes were overexpressed. Our study results showed differences between expression profiles of deoxyribonucleic acid repair genes in lung adenocarcinoma and malignant pleural mesothelioma cells. Based on our study results, we suggest that TREX1, PRKDC, and PMS1 genes may play a key role in the differential diagnosis of these two entities.

Germinal Immunogenetics predict treatment outcome for PD-1/PD-L1 checkpoint inhibitors

Background Checkpoint inhibitors bring marked benefits but only in a minority of patients and may also be associated with severe adverse events. Treatment outcome still cannot be faithfully predicted. The following study hypothesized that host genetics could be applied as predictive biomarkers for checkpoint inhibitor response and immune-related adverse events. We conducted a study based on germinal polymorphisms from genes coding for proteins involved in immune regulation. Methods Germinal DNA was obtained from advanced cancer patients treated with anti-PD-1/PD-L1 checkpoint inhibitors. DNA was genotyped using a custom panel of 166 single nucleotide polymorphisms covering 86 preselected immunogenetic-related genes. Computational analysis using a GTEX portal was made to determine potential expression Quantitative Trait Loci in tissues. Results Ninety-four consecutive patients were included. Objective response rate (complete or partial response) was significantly correlated to tumor microenvironment-related SNPs concerning CCL2, NOS3, IL1RN, IL12B, CXCR3 and IL6R genes. Toxicity were linked to target-related gene SNPs including UNG, IFNW1, CTLA4, PD-L1 and IFNL4 genes. The Area Under the ROC curve (AUC) was 0.81 (95% CI: 0.72-0.9) for response and 0.89 (95% CI: 0.76-1.00) for toxicity. In silico functionality exploring pointed rs4845618 (IL6R), rs10964859 (IFNW1) and rs3087243 (CTLA4) as potentially impacting gene expression. Conclusion These results strongly support a role for distinct immunogenetic-related gene SNPs able to predict efficacy and safety of anti-PD1/PD-L1 therapies. The results highlight the existence of patient-specific, germinal biomarkers able predict response to checkpoint inhibitor efficacy and, possibly, to predict treatment-related adverse events.

Uracil-DNA glycosylase UNG1 isoform variant supports class switch recombination and repairs nuclear genomic uracil.

UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

Abstract 1282: Activity of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT is associated with drug sensitivity in specific cancer subtypes

Abstract We used gene expression information and mutational signature analysis of cell lines from a diverse range of cancerous tissues to examine the activity of two members of the APOBEC family of cytidine deaminases, APOBEC3A and APOBEC3B, and three additional genes. APOBEC3B is known to increase the mutation load in many cancers, generating kataegis clusters of closely spaced, single strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested that APOBEC3A, REV1, UNG, and FHIT may also participate in hypermutation processes associated with APOBEC activity. We investigated how the activities of these five genes may affect the abundance of APOBEC-like signatures and whether they may be associated with sensitivity of cancer cells to treatment in different cancer categories. We analyzed several data sources for 1,408 cell lines from 26 cancer types available from the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC) resources. These data included whole exome sequencing (WES) data, microarray gene expression information, and drug sensitivity information. We examined correlations of the abundance of APOBEC-associated motifs and WES-wide mutation loads with APOBEC3A, APOBEC3B, REV1, UNG, and FHIT gene expression, as well as their association with cell line chemosensitivity to 255 antitumor drugs in multiple cancer subtypes. We were able to confirm several previously reported expression and chemosensitivity associations and found additional correlations, which may be clinically important. Sensitivity to JQ1, JNK inhibitors AS601245 and VIII, BMS-509744, bicalutamide, and several other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancer categories such as pancreatic, breast and non-small cell lung cancer cell lines. For example, we observed a strong negative correlation between APOBEC3A expression in pancreatic cell lines and sensitivity to JQ1, a BET inhibitor, which had been reported to inhibit pancreatic cancer cells in vitro and in vivo. In glioma cell lines, APOBEC3B expression was significantly negatively correlated with sensitivity to a CDK9 inhibitor THZ-2-49 and HSP90 inhibitors AUY922 and 17-AAG (tanespimycin). We observed a strong correlation between the combined length of kataegis clusters and chemoresistance to bicalutamide, a nonsteroidal antiandrogen drug, in breast cancer cell lines. Our findings suggest that associations of sensitivity to drug treatment with activities of APOBEC3A/B, REV1, UNG, and FHIT and with the rates of APOBEC-associated mutagenic processes may vary among different cancer categories. Citation Format: Suleyman Vural, Julia Krushkal, Richard Simon. Activity of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT is associated with drug sensitivity in specific cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1282.

Aging related changes in circulating reactive oxygen species (ROS) and protein carbonyls are indicative of liver oxidative injury.

Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense mechanisms, plays a major role in inducing oxidative damage and cellular impairment, resulting in a general decline of the physiological functions. The aim of this work was to evaluate age-related changes in circulating ROS levels and plasma protein carbonyls, in very young (2 months aged), young (8 months aged) and in middle age (15 months aged) F344 rats. In addition, the DNA oxidative marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the expression of the DNA repair enzymes APE1, OGG1 and UNG genes were also measured in the liver of these animals. We also determined whether systemic oxidative stress reflects oxidative injury at organ level. Our results demonstrate that the increase in circulating ROS and protein carbonyl content occurs as early as middle age. Moreover, increased 8-OHdG in the liver of 15-month-old rats was at least in part associated with a reduced DNA damage repairing capacity as suggested by the down-regulation of APE1 gene expression. In addition, we demonstrated for the first time, that plasma carbonyls and liver 8-OHdG are well correlated, suggesting that plasma protein carbonyls may be used as a surrogate marker of oxidative injury in target organs.

Chromatin Histone Modifying and DNA Repair Inhibition Enhances the Anti-Myeloma Activity of Melphalan

Repair of DNA and, thus, preservation of the genetic code are critical for normal cellular function. However, tumor cells use DNA repair pathways to develop resistance to chemotherapy. Therefore, inhibiting DNA repair may override this drug resistance. Herein, we further investigate the mechanistic basis of the link between DNA repair efficiency and response to anti-myeloma therapy and provide evidence that histone modifiers might prove efficacious when used in combination with DNA-damaging agents.We studied clonal bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed multiple myeloma (MM) patients (12F/14M; median age 60 years, range 42-66) who responded (n=17) or did not respond (n=9) to subsequent melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. Cells were ex vivotreated with melphalan alone or in the presence of histone deacetylase inhibitor HDACi) vorinostat or the Non-Homologous End-Joining (NHEJ) inhibitor SCR7 and the extent of the DNA damage formation/repair (monoadducts by using Southern blot analysis; interstrand crosslinks by using quantitative-PCR; double-strand breaks repair using immunofluorescence quantification of γH2AX foci by confocal microscopy), the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) and the local chromatin condensation were evaluated. Finally, the expression of a focused panel of 84 genes involved in DNA damage response pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also examined.Both BMPCs and PBMCs from responders to melphalan therapy showed more condensed chromatin structure and slower rates of DNA repair activity (P<0.0022) compared to non-responders. Moreover, apoptosis rates of both BMPCs and PBMCs were inversely correlated with individual DNA repair efficiency, being higher in responders’ cells compared to those of non-responders (P=0.0011). PBMCs from MM patients showed higher looseness of chromatin structure, increased DNA repair activity and higher apoptosis rates compared to healthy controls. Microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients’ responders to subsequent therapy compared to non-responders. Particularly, responders’ PBMCs showed upregulation of ATR, BLM, DDB1, EXO1, FANCA, MRE11A, MSH2, PCNA, RAD50, RAD51, RPA1, XPA, XPC, XRCC1 and TP53 genes and downregulation of ATM, CDC25C, CDKN1A, CHEK2, ERCC1, MPG, PNKP and UNG genes compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that perturbation in the molecular components of DNA damage response pathways plays an important role in the therapeutic action of the genotoxic drugs. Interestingly, co-treatment of cells with melphalan and the NHEJ inhibitor SCR7 significantly reduced the rates of DNA damage repair and increased melphalan sensitivity of both BMPCs and PBMCs. Moreover, co-treatment of BMPCs with melphalan and the HDACi vorinostat resulted in hyperacetylation of histone H4, increased DNA damage burden and higher apoptosis rates of these cells. On the other hand, co-treatment of PBMCs with melphalan in the presence of vorinostat did not significantly alter the melphalan-induced DNA damage burden and the apoptosis rates of these cells. Interestingly, using Western blot analysis, we found that vorinostat decreased the levels of critical DNA repair proteins (Rad50, Mre11, Ku70, Ku86, DNA-PKcs) in malignant BMPCs, while it did not suppressed these DNA proteins in PBMCs, a finding that can explain, in part, the selectivity of vorinostat in causing cell death in malignant cells at concentrations that cause little or no normal cell death.Collectively, responders to melphalan therapy are characterized by slower rates of DNA repair mechanisms, resulting in higher accumulation of the cytotoxic DNA lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. The enhancement of melphalan cytotoxicity by DNA repair modifiers offers a promising strategy toward treatment of MM and improvement of existing regimens. DisclosuresTerpos:Novartis: Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria.

Stress specific Escherichia coli biosensors based on gene promoters for toxicity monitoring

Target specific biosensor Escherichia coli strains have been engineered for studying the stress action. Four different recombinant E. coli biosensors were constructed by fusing one promoter each of ung, rseA, ibpA, and yggX gene in pET-21a::lacZ system, which contains very effective color-readout sensing system. The responses of four bacteria sensor strains, UNGLacZ, RSEALacZ, IBPALacZ, and YGGXLacZ, have been characterized under treatment with mitomycin C (MMC), phenol, ethanol and hydrogen peroxide (H2O2), respectively. UNGLacZ responded strongly only to MMC (DNA damaging chemical), whereas no response has been observed with phenol, which is a membrane damage chemical. However, RSEALacZ gave significant response with phenol. In addition, ethanol and hydrogen peroxide caused dose-dependent induction from IBPALacZ and YGGXLacZ, respectively. Thus, the results of the present study demonstrate that four bacterial strains show significant and different responses based on various stress defense mechanisms, which these genes originally possessed in E. coli. The four bacterial biosensors can be potentially used in environmental Point-of-Care Testing (ePOCT) leading to quick, easy and early detection of toxic contaminants locally as well as resulting in real-time measurements.

Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.

Evaluation of a Novel Missense Activation-Induced Deaminase AID Mutation in a Child with Hyper IgM Syndrome: Is it a Pathogenic Mutation?

Hyper IgM Syndrome (HIGM) is a primary immunodeficiency associated with defects of CD40, CD40L, AID and UNG genes. In the case of a novel mutation, genetic testing requires secondary confirmation such as functional studies to establish a link between the clinical phenotype and the mutation identified.

Surfactant Protein A (SP-A) Reduces Human Rhinovirus 16 (RV16)-Induced Inflammatory Responses In Bronchial Epithelial Cells and Inhibits Viral Replication In H1-HeLa Cells

FEBRUARY 2014 AB70 Abstracts S A T U R D A Y 248 Evaluation of a Novel Missense Activation-Induced Deaminase AID Mutation in a Child with Hyper IgM Syndrome: Is it a Pathogenic Mutation? Dr. Ottavia M. Delmonte, MD, Dr. Feilong Meng, PhD, Dr. Frederick Alt, PhD, Dr. Luigi D. Notarangelo, MD, Dr. Jolan E. Walter, MD, PhD; Pediatric Residency Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA, Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine and Immune Disease Institute, Boston Children’s Hospital, Department of Genetcis, Harvard Medical School, Boston, MA, Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine and Immune Disease Institute, Boston Children’s Hospital, Department of Genetics, Harvard Medical School, Boston, MA, Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA; The Manton Center for Orphan Disease Research, Boston Children’s Hospital, Boston, MA, Division of Allergy/Immunology, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA. RATIONALE: Hyper IgM Syndrome (HIGM) is a primary immunodeficiency associated with defects of CD40, CD40L, AID and UNG genes. In the case of a novel mutation, genetic testing requires secondary confirmation such as functional studies to establish a link between the clinical phenotype and the mutation identified. METHODS: Genetic screening of a 4-year-old boy with clinical features of HIGM Syndrome resulted in a novel missense mutation in the AID gene (p.Ala132Pro). The mutation was located away from the core catalytic domain, but in an area highly conserved among species. The effect of the mutation on AID expression or function was unclear. Testing was undertaken to connect the novel mutation to an actual defect in AID, in vivo and in vitro. In vitro studies included compliment class switch recombination (CSR) assay in AICDA-deficient murine B cells andWestern blot to detect protein stability. AID function in vivo was assessed by IgG level and switched memory B cell fraction. RESULTS: Functional studies confirmed that the mouse AID A132P mutant cannot support CSR in AICDA-deficient murine B cells with retroviral-delivered AID mutant proteins. The AID mutant results in a dysfunctional unstable protein which cannot be detected with Western blot, suggesting that this conserved amino acid could be involved in protein folding and stability. In vivo, the very low amount of AID resulted in low IgG level (<7 mg/dL) and a low fraction of class switched memory B cells (<2%). CONCLUSIONS: Link should be established with functional studies for novel mutations, especially in case of missense mutations.

ASSOCIATION BETWEEN POLYMORPHISM OF THE DNA REPAIR SMUG1 AND UNG GENES AND AGE-RELATED MACULAR DEGENERATION

To investigate the association between the g.4235T>C (rs2337395) polymorphism of the UNG gene and the c.-31A>G (rs3087404) polymorphism of the SMUG1 gene and the risk of age-related macular degeneration (AMD), as well as modulation of this association by some environmental and lifestyle factors. Overall, 272 AMD patients and 105 control subjects were enrolled in this study. Both polymorphisms were genotyped by restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP). The C/C genotype of the g.4235T>C polymorphism of the UNG gene was associated with an increased risk of dry AMD (odds ratio, 2.54), whereas the T/T genotype of this polymorphism decreased such risk (odds ratio, 0.41). The presence of the T allele of the g.4235T>C polymorphism and the A allele of the c.-31A>G polymorphism of the SMUG1 gene (odds ratio, 2.17 and 2.18, respectively) was associated with an increased risk of AMD severity, expressed by the comparison of the frequencies of genotypes in the group of patients with wet AMD versus those with dry AMD. Conversely, the C/C genotype of the g.4235T>C polymorphism, the G/G genotype of the c.-31A>G polymorphism, and the C/C-G/G combined genotype of both polymorphisms had a protective effect (odds ratio, 0.48, 0.46, and 0.18; respectively). The results obtained suggest the potential role of the g.4235T>C and the c.-31A>G polymorphisms in AMD pathogenesis.

Multiple microRNAs may regulate the DNA repair enzyme uracil-DNA glycosylase

Human nuclear uracil-DNA glycosylase UNG2 is essential for post-replicative repair of uracil in DNA, and UNG2 protein and mRNA levels rapidly decline in G2/M phase. Previous work has demonstrated regulation of UNG2 at the transcriptional level, as well as by protein phosphorylation and ubiquitylation. UNG2 mRNA, encoded by the UNG gene, contains a long 3′untranslated region (3′UTR) of previously unknown function. Here, we demonstrate that several conserved regions in the 3′UTR are potential seed sites for microRNAs (miRNAs), such as miR-16, miR-34c, and miR-199a. Our results show that these miRNAs down-regulate UNG activity, UNG mRNA, and UNG protein levels. Down-regulation was dependent on the 3′UTR, indicating that the miRNAs directly target the conserved seed sites in the 3′UTR. These results add miRNAs as a new modality to UNG's increasing list of complex regulatory mechanisms.

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.

Human endonuclease V as a repair enzyme for DNA deamination

The human endonuclease V gene is located in chromosome 17q25.3 and encodes a 282 amino acid protein that shares about 30% sequence identity with bacterial endonuclease V. This study reports biochemical properties of human endonuclease V with respect to repair of deaminated base lesions. Using soluble proteins fused to thioredoxin at the N-terminus, we determined repair activities of human endonuclease V on deoxyinosine (I)-, deoxyxanthosine (X)-, deoxyoxanosine (O)- and deoxyuridine (U)-containing DNA. Human endonuclease V is most active with deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Endonuclease activities on deoxyuridine and deoxyoxanosine were not detected. The endonuclease activity on deoxyinosine-containing DNA follows the order of single-stranded I>G/I>T/I>A/I>C/I. The preference of the catalytic activity correlates with the binding affinity of these deoxyinosine-containing DNAs. Mg2+ and to a much less extent, Mn2+, Ni2+, Co2+ can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells deficient in nfi, mug and ung genes caused three-fold reduction in mutation frequency. This is the first report of deaminated base repair activity for human endonuclease V. The relationship between the endonuclease activity and deaminated deoxyadenosine (deoxyinosine) repair is discussed.

Abstract A100: Pemetrexed-induced uracil accumulation is an importatn source of DNA damage in UNG deficient cells, contributing to hematologic toxicity.

Abstract Pemetrexed is a multi-target antifolate that has proven activity against several solid tumors. Dose limiting myelosuppression and acquired resistance are major challenges to pemetrexed and antifolate chemotherapy regimens. We have previously shown that resistance to pemetrexed in human cancer cells can be overcome via inhibition of DNA base excision repair (BER), the major pathway responsible for the resolution of uracil in DNA. To understand the implications of UNG deficiency in vivo, we have investigated the impact of UNG deficiency on whole animal response to pemetrexed. Here, we illustrate that pemetrexed treatment induces more significant reduction in bone marrow cell numbers and relatively low peripheral lymphocyte counts in UNG−/− mice compared to UNG+/+ littermates. Compared to wild-type cells, UNG−/− MEFs also show reduced viability and increased cell death in response to pemetrexed. Hematopoietic cell sensitivity was mirrored by increased pemetrexed cell killing in UNG mutant human lymphoblastoid cells, derived from patients with biallelic inactivating mutations in the UNG gene. Cellular sensitivity is characterized by reduced nucleotide incorporation, arrest in S-phase of the cell cycle, and induction of apoptosis. Pemetrexed treatment of UNG−/− cells also causes robust induction of γ-H2AX, a surrogate marker of DNA double strand breaks. Through γ-H2AX ChIP-sequencing we provide the first direct evidence that DNA double strand breaks are located specifically at AT(U) rich sequences, directly implicating uracil accumulation in the formation of double strand breaks following pemetrexed treatment. UNG deficient cells showed no differential sensitivity to cisplatin or temozolomide, two drugs unrelated to uracil accumulation. Moreover, through thymidine supplementation, we were able to partially reverse the pemetrexed-mediated induction of DNA damage markers, including γ-H2AX and protect UNG−/− cells from cell death, further supporting a uracil-mediated mechanism of cytotoxicity in UNG−/− cells. In sum, these data suggest that the accumulation of uracil in the DNA of hematopoietic cells drives myelosuppression and lymphocyte toxicity. Mechanistically, our data support a previously underappreciated role for uracil accumulation, UNG activity and the BER pathway in the determination of cellular sensitivity to pemetrexed and suggest UNG expression may be an important predictor of adverse events, such as myelosuppression in individuals receiving pemetrexed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A100.

Study of patients with Hyper-IgM type IV phenotype who recovered spontaneously during late childhood and review of the literature

Hyper-IgM syndromes are characterized by normal or elevated serum IgM levels with the absence or reduced levels of other immunoglobulins. There are some patients with defective class-switch recombination (CSR) who do not have CD40L, CD40, AID, and UNG defects. The aim of this study is to determine the B-cell functions of patients with Hyper-IgM type 4 phenotype. Ten patients (seven males and three females) 84.2 ± 16.5months of age with initial low serum IgG and IgA and high or normal IgM levels were included. Clinically, 50% had recurrent upper respiratory tract, 10% urinary tract, 10% lower respiratory tract infections, and 30% had mixed type infections. Lymphoid hyperplasia, overt autoimmune manifestations, or malignancy was not noted. Seven of 10 patients were studied twice; at the age of 34.2 ± 13.7 and at 86.6 ± 12.3months. Absolute lymphocyte counts and lymphocyte subsets were normal in all cases. All of them had normal expression of CD40 on B cells and CD40L on activated T cells for males. At first examination, all patients had normal in vitro sCD40L+rIL-4-induced B cell proliferation response and somatic hypermutation but CSR towards IgE was absent. AID and UNG genes did not show any abnormalities. All showed improvement in both clinical findings and Ig levels during the follow-up period of 55.8 ± 14.8months. Ages for normalization of IgG and IgA were 68.2 ± 8.7 and 70.2 ± 21.6months, respectively. During the second evaluation: In vitro sCD40L+rIL-4-induced B-cell proliferation was normal in all cases, whereas CSR was still abnormal in five of eight patients. Two of the patients had an increase in in vitro CSR response but still low IgG2 subclass levels. Three patients with initially absent in vitro CSR response also normalized. Clinical manifestations and immunoglobulin levels of the patients with Hyper-IgM type 4 phenotype recovered in late childhood at about 6years of age. There was a transient CSR defect which was not observed in cases with transient hypogammaglobulinemia of infancy. Detection of a non-AID or non-UNG associated CSR defect in infancy should be confirmed later on since spontaneous recovery may occur.