Lost in the Crowd: How Does Human 8-Oxoguanine DNA Glycosylase 1 (OGG1) Find 8-Oxoguanine in the Genome?

The most frequent DNA lesion resulting from an oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a premutagenic base modification due to its capacity to pair with adenine. Thus, the repair of 8-oxoG is critical for the preservation of the genetic information. Nowadays, 8-oxoG is also considered as an oxidative stress-sensor with a putative role in transcription regulation. In mammalian cells, the modified base is excised by the 8-oxoguanine DNA glycosylase (OGG1), initiating the base excision repair (BER) pathway. OGG1 confronts the massive challenge that is finding rare occurrences of 8-oxoG among a million-fold excess of normal guanines. Here, we review the current knowledge on the search and discrimination mechanisms employed by OGG1 to find its substrate in the genome. While there is considerable data from in vitro experiments, much less is known on how OGG1 is recruited to chromatin and scans the genome within the cellular nucleus. Based on what is known of the strategies used by proteins searching for rare genomic targets, we discuss the possible scenarios allowing the efficient detection of 8-oxoG by OGG1.

AUTACs: Cargo-Specific Degraders Using Selective Autophagy.

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene.

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5' untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5'UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

Anhydrous ß-guanine crystals in a marine dinoflagellate: Structure and suggested function.

Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo- FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the ß-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.

Photonic crystals cause active colour change in chameleons.

Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study.

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.

8-Oxoguanine causes neurodegeneration during MUTYH-mediated DNA base excision repair.

8-Oxoguanine (8-oxoG), a common DNA lesion caused by reactive oxygen species, is associated with carcinogenesis and neurodegeneration. Although the mechanism by which 8-oxoG causes carcinogenesis is well understood, the mechanism by which it causes neurodegeneration is unknown. Here, we report that neurodegeneration is triggered by MUTYH-mediated excision repair of 8-oxoG-paired adenine. Mutant mice lacking 8-oxo-2'-deoxyguanosine triphosphate-depleting (8-oxo-dGTP-depleting) MTH1 and/or 8-oxoG-excising OGG1 exhibited severe striatal neurodegeneration, whereas mutant mice lacking MUTYH or OGG1/MUTYH were resistant to neurodegeneration under conditions of oxidative stress. These results indicate that OGG1 and MTH1 are protective, while MUTYH promotes neurodegeneration. We observed that 8-oxoG accumulated in the mitochondrial DNA of neurons and caused calpain-dependent neuronal loss, while delayed nuclear accumulation of 8-oxoG in microglia resulted in PARP-dependent activation of apoptosis-inducing factor and exacerbated microgliosis. These results revealed that neurodegeneration is a complex process caused by 8-oxoG accumulation in the genomes of neurons and microglia. Different signaling pathways were triggered by the accumulation of single-strand breaks in each type of DNA generated during base excision repair initiated by MUTYH, suggesting that suppression of MUTYH may protect the brain under conditions of oxidative stress.

Association between TNF-α promoter -308A/G polymorphism and tardive dyskinesian Chinese Han patients with schizophrenia.

OBJECTIVE: Previous studies have indicated that the immune may be involved in the pathogenesis of tardive dyskinesia (TD). Some genetic polymorphisms in the human leukocyte antigen (HLA) I and II regions have been associated with TD, and the tumor necrosis factor-α (TNF-α) gene is located in the HLA III region. TNF-α levels in the striatum significantly increased in haloperidol-induced TD in rats. The TNF-α gene -308A/G single nucleotide polymorphism (SNP) has been shown to directly influence TNF-α expression. The genetic association between the TNF-α gene -308A/G SNP and TD is unclear. The present study investigated whether this variation is associated with clinical phenotypes and TD in schizophrenia in a genetically homogeneous northern Chinese Han population. METHODS: We genotyped the TNF-α gene -308A/G SNP in patients with schizophrenia with TD (n=350) and without TD (n=410). The Abnormal Involuntary Movement Scale (AIMS) and Positive and Negative Syndrome Scale (PANSS) were used to assess the severity of TD and psychopathology of schizophrenia, respectively. RESULTS: The allele and genotype frequencies did not significantly differ between patients with schizophrenia with and without TD (p>0.05). No significant difference was found in the total AIMS score between the genotypes (p>0.05). However, the PANSS negative symptom subscore was associated with risk for TD (p=0.004), and a significant difference was found in total AIMS score between the genotypes in TD patients (p=0.013). CONCLUSION: The TNF-α gene -308A/G polymorphism does not appear to play a major role in the susceptibility to TD in patients with schizophrenia in a northern Chinese Han population. However this polymorphism may play a role in the TD severity.

Examination of the telomere G-overhang structure in Trypanosoma brucei.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

The role of iNOS-mediated DNA damage in infection- and asbestos-induced carcinogenesis.

Chronic inflammation contributes to a substantial part of environmental carcinogenesis. Various infectious diseases and physical, chemical, and immunological factors participate in inflammation-related carcinogenesis. Under inflammatory conditions, reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells, and resulting DNA damage may participate in carcinogenesis. 8-Nitroguanine is a mutagenic DNA lesion formed during chronic inflammation. We performed immunohistochemical analysis, and demonstrated that 8-nitroguanine was formed at the sites of carcinogenesis in animal models and patients with various cancer-prone infectious and inflammatory diseases, caused by parasites, viruses, and asbestos exposure. In asbestos-exposed mice, 8-nitroguanine was formed in bronchial epithelial cells, and it is noteworthy that crocidolite induced significantly more intense 8-nitroguanine formation than chrysotile, inconsistent with their carcinogenic potentials. On the basis of these findings, we have proposed that 8-nitroguanine could be a potential biomarker to evaluate the risk of inflammation-related carcinogenesis.

Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system.

OBJECTIVE: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the "MEGS capacity." Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A-->G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome. METHODS: In muscle tissue of 24 patients with the 3243A-->G mutation, we investigated the MEGS capacity, the respiratory chain enzymatic activities, and the 3243A-->G mutation load. To exclude coinciding mutations, we sequenced all 22 mitochondrial transfer RNA genes in the patients, if possible. RESULTS: We found highly significant differences between patients and control subjects with respect to the MEGS capacity and complex I, III, and IV activities. MEGS-related measurements correlated considerably better with the mutation load than respiratory chain enzyme activities. We found no additional mutations in the mitochondrial transfer RNA genes of the patients. INTERPRETATION: The results show that MEGS capacity has a greater sensitivity than respiratory chain enzymatic activities for detection of subtle mitochondrial dysfunction. This is important in the workup of patients with rare or new mitochondrial DNA mutations, and with low mutation loads. In these cases we suggest to determine the MEGS capacity.

Skin color in fish and humans: impacts on science and society.

As genetic model systems, fish have played a key role in our understanding of a wide range of biological processes, including vertebrate pigmentation. In this review, we focus on one aspect of pigmentation, skin pigmentation, which has been of momentous importance in human history. Two fish models, medaka and zebrafish, played important roles in demystifying skin color and, by extension, the concept of "race." Related thinking has the potential to make two additional contributions to human welfare. Fish can be used to validate gene candidates from genome-wide association studies (GWAS) in what has been called "Systems Genetics." Because fish are familiar vertebrates, and share genetic mechanisms of skin color with humans, they also have outstanding potential as an educational tool-to "demystify" race, to increase public understanding of the role of model systems and evolution in science, and to enhance appreciation of both genetic and environmental factors that impact human health and society.

G>C SNP of thymidylate synthase with respect to colorectal cancer.

Several studies indicate that low thymidylate synthase (TS) protein levels in tumor and normal tissues of colorectal cancer patients are associated with better clinical response to fluorouracil-based chemotherapy and higher risk of toxicity. However, no correlation or even reverse correlation has also been reported. These conflicting results may be partly due to the methodological limitations of the immunohistochemical techniques generally used to quantify thymidylate synthase expression. In this sense, a genetic approach aiming at determining the influence of the TS gene polymorphisms on clinical outcome seems more appealing. So far three polymorphisms have been identified and studied in the TYMS gene: the variable number of 28-bp tandem repeats (2R or 3R) in the 5 UTR; the G>C substitution at the 12th nucleotide in the second repeat of the 3R allele (3RG>3RC) and the 6-bp deletion in the 3 UTR (+6bp/-6bp 3 UTR). In vitro studies indicate that each of these polymorphisms can influence thymidylate synthase expression. In particular, the G>C SNP, which alters the E-box sequence binding an upstream stimulatory factor (USF-1), seems more important than the variable number of tandem repeats in determining TS gene expression in that the 3RC allele has a reduced translational activity compared with the 3RG allele, while showing the same activity as the 2R allele. In contrast with the in vitro findings, the clinical studies in colorectal patients failed to find a consistent relationship between the G>C polymorphism and clinical outcome measures (response, survival or toxicity). This discrepancy may be due to methodological heterogeneities amongst the studies, including genotyping in normal or tumor tissues, loss of heterozygosity in tumor cells not evaluated, variable doses and schedules of fluorouracil-based therapy, and variable tumor stage. The complexity of TYMS gene regulation, and the possibility that other polymorphisms may contribute to fluorouracil response, call for further studies before TYMS genotyping can be used in clinical practice to select colorectal cancer patients who are most likely to benefit from chemotherapy.

An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein.

Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif.

Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II.

CONTEXT: RGS2 (regulators of G-protein signaling) is a negative regulator of Galphaq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. OBJECTIVE: To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. METHODS: PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3' untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). RESULTS: RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. CONCLUSION: Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion.

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG*C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

Guanine-adenine bias: a general property of retroid viruses that is unrelated to host-induced hypermutation.

The recently discovered mammalian enzymes, APOBEC3G and 3F, induce guanine-to-adenine hypermutation in retroviruses. However, the preference of adenine over guanine in retroviral codon usage is not correlated with the presence or absence of APOBEC3G or its viral inhibitor (Vif), and its pattern does not reflect the biochemical properties of APOBEC3G action. The guanine-adenine bias of retroviruses is thus probably not a result of host-induced mutational pressure, but rather reflects a general predisposition associated with reverse transcription.

Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.

AlkB repairs 1-alkyladenine and 3-methylcytosine lesions in DNA by directly reversing the base damage. Although repair studies with randomly alkylated substrates have been performed, the miscoding nature of these and related individually alkylated bases and the suppression of mutagenesis by AlkB within cells have not yet been explored. Here, we address the miscoding potential of 1-methyldeoxyadenosine (m1A), 3-methyldeoxycytidine (m3C), 3-ethyldeoxycytidine (e3C), 1-methyldeoxyguanosine (m1G), and 3-methyldeoxythymidine (m3T) by synthesizing single-stranded vectors containing each alkylated base, followed by vector passage through Escherichia coli. In SOS(-), AlkB-deficient cells, m1A was only 1% mutagenic; however, m3C and e3C were 30% mutagenic, rising to 70% in SOS(+) cells. In contrast, the mutagenicity of m1G and m3T in AlkB(-) cells dropped slightly when SOS polymerases were expressed (m1G from 80% to 66% and m3T from 60% to 53%). Mutagenicity was abrogated for m1A, m3C, and e3C in wild-type (AlkB(+)) cells, whereas m3T mutagenicity was only partially reduced. Remarkably, m1G mutagenicity was also eliminated in AlkB(+) cells, establishing it as a natural AlkB substrate. All lesions were blocks to replication in AlkB-deficient cells. The m1A, m3C, and e3C blockades were completely removed in wild-type cells; the m1G blockade was partially removed and that for m3T was unaffected by the presence of AlkB. All lesions demonstrated enhanced bypass when SOS polymerases were induced. This work provides direct evidence that AlkB suppresses both genotoxicity and mutagenesis by physiologically realistic low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damage in vivo.