Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma.

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.

8-Oxo-guanine DNA damage induces transcription errors by escaping two distinct fidelity control checkpoints of RNA polymerase II.

RNA polymerase II (Pol II) has an intrinsic fidelity control mechanism to maintain faithful genetic information transfer during transcription. 8-Oxo-guanine (8OG), a commonly occurring damaged guanine base, promotes misincorporation of adenine into the RNA strand. Recent structural work has shown that adenine can pair with the syn conformation of 8OG directly upstream of the Pol II active site. However, it remains unknown how 8OG is accommodated in the active site as a template base for the incoming ATP. Here, we used molecular dynamics (MD) simulations to investigate two consecutive steps that may contribute to the adenine misincorporation by Pol II. First, the mismatch is located in the active site, contributing to initial incorporation of adenine. Second, the mismatch is in the adjacent upstream position, contributing to extension from the mismatched bp. These results are supported by an in vitro transcription assay, confirming that 8OG can induce adenine misincorporation. Our simulations further suggest that 8OG forms a stable bp with the mismatched adenine in both the active site and the adjacent upstream position. This stability predominantly originates from hydrogen bonding between the mismatched adenine and 8OG in a noncanonical syn conformation. Interestingly, we also found that an unstable bp present directly upstream of the active site, such as adenine paired with 8OG in the canonical anti conformation, largely disrupts the stability of the active site. Our findings have uncovered two main factors contributing to how 8OG induces transcriptional errors and escapes Pol II transcriptional fidelity control checkpoints.

Detection of DNA replication errors and 8-oxo-dGTP-mediated mutations in E. coli by Duplex DNA Sequencing.

Mutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107 base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations in E. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using a mutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10-7 to 10-8 depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurred in vivo. In contrast, bona-fide in vivo mutations were identified for DNA from both the mutL and mutT strains, as indicated by specific increases in base substitutions that are fully consistent with their established in vivo roles. Notably, the data reproduce the specific context effects of in vivo mutations as well as the leading vs. lagging strand bias among DNA replication errors.

Quantifying telomere transcripts as tool to improve risk assessment for genetic instability and genotoxicity.

Telomere repeat-containing RNAs (TERRA) are transcribed from telomeres as long non-coding RNAs and are part of the telomere structure with protective function. The genetic stability of cells requires telomeric repeats at the ends of chromosomes. Maintenance of telomere length (TL) is essential for proliferative capacity and chromosomal integrity. In contrast, telomere shortening is a recognized risk factor for carcinogenesis and a biomarker of aging due to the cumulative effects of environmental exposures and life experiences such as trauma or stress. In this context, telomere repeats are lost due to cell proliferation, but are also susceptible to stress factors including reactive oxygen species (ROS) inducing oxidative base damage. Quantitative PCR (qPCR) of genomic DNA is an established method to analyze TL as a tool to detect genotoxic events. That same qPCR method can be applied to RNA converted into cDNA to quantify TERRA as a useful tool to perform high-throughput screenings. This short review summarizes relevant qPCR studies using both TL and TERRA quantification, provides an overall view of the molecular mechanisms of telomere protection against ROS by TERRA, and summarizes the presented studies comparing the results at DNA and RNA levels, which indicate that fluctuations at transcript level might reflect a short-term response. Therefore, we conclude that performing both of these measurements together will improve genotoxicity studies.

Optimization of N-Piperidinyl-Benzimidazolone Derivatives as Potent and Selective Inhibitors of 8-Oxo-Guanine DNA Glycosylase 1.

8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : C→T : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.

Targeted Formation of 8-Oxoguanine in Telomeres.

Mammalian telomeres are guanine-rich sequences which cap the ends of linear chromosomes. While recognized as sites sensitive to oxidative stress, studies on the consequences of oxidative damage to telomeres have been primarily limited to experimental conditions which cause oxidative damage throughout the whole genome and cell. We developed a chemoptogenetic tool (FAP-mCER-TRF1) to specifically induce singlet oxygen at telomeres, resulting in the formation of the common oxidative lesion 8-oxo-guanine. Here, we describe this tool and detail how to generate cell lines which express FAP-mCER-TRF1 at telomeres and verify the formation of 8-oxo-guanine.

Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine Bypass by Family X DNA Polymerases.

8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols ß, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles-the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.

Almond Skin Extracts and Chlorogenic Acid Delay Chronological Aging and Enhanced Oxidative Stress Response in Yeast.

Almond (Prunus dulcis (Mill.) D.A.Webb) is one of the largest nut crops in the world. Recently, phenolic compounds, mostly stored in almond skin, have been associated with much of the health-promoting behavior associated with their intake. The almond skin enriched fraction obtained from cold-pressed oil residues of the endemic Moroccan Beldi ecotypes is particularly rich in chlorogenic acid. In this study, both almond skin extract (AE) and chlorogenic acid (CHL) supplements, similar to traditional positive control resveratrol, significantly increased the chronological life-span of yeast compared to the untreated group. Our results showed that AE and CHL significantly reduced the production of reactive oxygen and nitrogen species (ROS/RNS), most likely due to their ability to maintain mitochondrial function during aging, as indicated by the maintenance of normal mitochondrial membrane potential in treated groups. This may be associated with the observed activation of the anti-oxidative stress response in treated yeast, which results in activation at both gene expression and enzymatic activity levels for SOD2 and SIR2, the latter being an upstream inducer of SOD2 expression. Interestingly, the differential gene expression induction of mitochondrial SOD2 gene at the expense of the cytosolic SOD1 gene confirms the key role of mitochondrial function in this regulation. Furthermore, AE and CHL have contributed to the survival of yeast under UV-C-induced oxidative stress, by reducing the development of ROS/RNS, resulting in a significant reduction in cellular oxidative damage, as evidenced by decreased membrane lipid peroxidation, protein carbonyl content and 8-oxo-guanine formation in DNA. Together, these results demonstrate the interest of AE and CHL as new regulators in the chronological life-span and control of the oxidative stress response of yeast.

Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis.

Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.

Transcriptional Fidelity of Mitochondrial RNA Polymerase RpoTm from Arabidopsis thaliana.

Fidelity of RNA synthesis is essential for the faithful transfer of information from DNA to RNA. A comprehensive analysis of the nucleotide selectivity by the mitochondrial RNA polymerase (RNAP) RpoTm, from Arabidopsis thaliana, has been carried out. The kinetic parameters for the incorporation of cognate, noncognate, and oxidized bases have been determined. The results establish high fidelity of mitochondrial transcription resembling those of replicative polymerases in the absence of repair. In addition, RpoTm incorporates oxidized nucleotides with similar efficiency compared with mismatches and is capable of extending the RNA beyond the insertion of the oxidized base. Furthermore, lesion bypass study on RpoTm demonstrates that the enzyme bypasses 8-oxo-guanine by insertion of adenine leading to C to A mutations in RNA. Homology modeling of RpoTm elongation complex allows delineation of the residues necessary for stabilizing the incoming NTP substrate and for posing the template nucleotide residue. Substitution of these residues leads to compromise in the activity of the enzyme corroborating their importance in RNA synthesis. Comparison of the data with T7 RNAPs indicates that low efficiency of misincorporation is a universal strategy used by single-subunit RNAPs for maintaining high fidelity in the absence of proofreading and repair activity in mitochondria.

Mapping three guanine oxidation products along DNA following exposure to three types of reactive oxygen species.

Reactive oxygen and nitrogen species generated during respiration, inflammation, and immune response can damage cellular DNA, contributing to aging, cancer, and neurodegeneration. The ability of oxidized DNA bases to interfere with DNA replication and transcription is strongly influenced by their chemical structures and locations within the genome. In the present work, we examined the influence of local DNA sequence context, DNA secondary structure, and oxidant identity on the efficiency and the chemistry of guanine oxidation in the context of the Kras protooncogene. A novel isotope labeling strategy developed in our laboratory was used to accurately map the formation of 2,2-diamino-4-[(2-deoxy-ß-D-erythropentofuranosyl)amino]- 5(2 H)-oxazolone (Z), 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 8-nitroguanine (8-NO2-G) lesions along DNA duplexes following photooxidation in the presence of riboflavin, treatment with nitrosoperoxycarbonate, and oxidation in the presence of hydroxyl radicals. Riboflavin-mediated photooxidation preferentially induced OG lesions at 5' guanines within GG repeats, while treatment with nitrosoperoxycarbonate targeted 3'-guanines within GG and AG dinucleotides. Little sequence selectivity was observed following hydroxyl radical-mediated oxidation. However, Z and 8-NO2-G adducts were overproduced at duplex ends, irrespective of oxidant identity. Overall, our results indicate that the patterns of Z, OG, and 8-NO2-G adduct formation in the genome are distinct and are influenced by oxidant identity and the secondary structure of DNA.

MutT from the fish pathogen Aliivibrio salmonicida is a cold-active nucleotide-pool sanitization enzyme with unexpectedly high thermostability.

Upon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism's first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well-functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide-pool sanitization enzyme, which degrades 8-oxo-dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogen Aliivibrio salmonicida (AsMutT) and the human pathogen Vibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold-adapted properties of AsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8-oxo-dGTP substrate, and that AsMutT has a higher catalytic efficiency than VcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (E a) for AsMutT compared to VcMutT, and differential scanning calorimetry experiments showed that AsMutT displayed an unexpected higher melting temperature (T m) value than VcMutT. A comparative analysis of the crystal structure of VcMutT, determined to 2.42 Å resolution, and homology models of AsMutT indicate that three unique Gly residues in loops of VcMutT, and additional long range ion-pairs in AsMutT could explain the difference in temperature stability of the two enzymes. We conclude that AsMutT is a stable, cold-active enzyme with high catalytic efficiency and reduced E a, compared to the mesophilic VcMutT.

MUTYH DNA glycosylase: the rationale for removing undamaged bases from the DNA.

Maintenance of genetic stability is crucial for all organisms in order to avoid the onset of deleterious diseases such as cancer. One of the many proveniences of DNA base damage in mammalian cells is oxidative stress, arising from a variety of endogenous and exogenous sources, generating highly mutagenic oxidative DNA lesions. One of the best characterized oxidative DNA lesion is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can give rise to base substitution mutations (also known as point mutations). This mutagenicity is due to the miscoding potential of 8-oxo-G that instructs most DNA polymerases (pols) to preferentially insert an Adenine (A) opposite 8-oxo-G instead of the appropriate Cytosine (C). If left unrepaired, such A:8-oxo-G mispairs can give rise to CG→AT transversion mutations. A:8-oxo-G mispairs are proficiently recognized by the MutY glycosylase homologue (MUTYH). MUTYH can remove the mispaired A from an A:8-oxo-G, giving way to the canonical base-excision repair (BER) that ultimately restores undamaged Guanine (G). The importance of this MUTYH-initiated pathway is illustrated by the fact that biallelic mutations in the MUTYH gene are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). In this review, we will focus on MUTYH, from its discovery to the most recent data regarding its cellular roles and interaction partners. We discuss the involvement of the MUTYH protein in the A:8-oxo-G BER pathway acting together with pol λ, the pol that can faithfully incorporate C opposite 8-oxo-G and thus bypass this lesion in a correct manner. We also outline the current knowledge about the regulation of MUTYH itself and the A:8-oxo-G repair pathway by posttranslational modifications (PTM). Finally, to achieve a clearer overview of the literature, we will briefly touch on the rather confusing MUTYH nomenclature. In short, MUTYH is a unique DNA glycosylase that catalyzes the excision of an undamaged base from DNA.