Label-free fluorescence detection of human 8-oxoguanine DNA glycosylase activity amplified by target-induced rolling circle amplification.

BACKGROUND: Human 8-oxoG DNA glycosylase 1 (hOGG1) is one of the important members of DNA glycosylase for Base excision repair (BER), the abnormal activity of which can lead to the failure of BER and the appearance of various diseases, such as breast cancer, bladder cancer, Parkinson's disease and lung cancer. Therefore, it is important to detect the activity of hOGG1. However, traditional detection methods suffer from time consuming, complicated operation, high false positive results and low sensitivity. Thus, it remains a challenge to develop simple and sensitive hOGG1 analysis strategies to facilitate early diagnosis and treatment of the relative disease. RESULTS: A target-induced rolling circle amplification (TIRCA) strategy for label-free fluorescence detection of hOGG1 activity was proposed with high sensitivity and specificity. The TIRCA strategy was constructed by a hairpin probe (HP) containing 8-oxoG site and a primer probe (PP). In the presence of hOGG1, the HP transformed into dumbbell DNA probe (DDP) after the 8-oxoG site of which was removed. Then the DDP formed closed circular dumbbell probe (CCDP) by ligase. CCDP could be used as amplification template of RCA to trigger RCA. The RCA products containing repeated G4 sequences could combine with ThT to produce enhanced fluorescence, achieving label-free fluorescence sensing of hOGG1. Given the high amplification efficiency of RCA and the high fluorescence quantum yield of the G4/ThT, the proposed TIRCA achieved highly sensitive measurement of hOGG1 activity with a detection limit of 0.00143 U/mL. The TIRCA strategy also exhibited excellent specificity for hOGG1 analysis over other interference enzymes. SIGNIFICANCE: This novel TIRCA strategy demonstrates high sensitivity and high specificity for the detection of hOGG1, which has also been successfully used for the screening of inhibitors and the analysis of hOGG1 in real samples. We believe that this TIRCA strategy provides new insight into the use of the isothermal nucleic acid amplification as a useful tool for hOGG1 detection and will play an important role in disease early diagnosis and treatment.

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase.

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Šresolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P21212 enabled the successful modeling of 96% of residues in the asymmetric unit, with final Rwork and Rfree values of 0.199 and 0.229, respectively.

Structural and biochemical insights into NEIL2's preference for abasic sites.

Cellular DNA is subject to damage from a multitude of sources and repair or bypass of sites of damage utilize an array of context or cell cycle dependent systems. The recognition and removal of oxidatively damaged bases is the task of DNA glycosylases from the base excision repair pathway utilizing two structural families that excise base lesions in a wide range of DNA contexts including duplex, single-stranded and bubble structures arising during transcription. The mammalian NEIL2 glycosylase of the Fpg/Nei family excises lesions from each of these DNA contexts favoring the latter two with a preference for oxidized cytosine products and abasic sites. We have determined the first liganded crystal structure of mammalian NEIL2 in complex with an abasic site analog containing DNA duplex at 2.08 Å resolution. Comparison to the unliganded structure revealed a large interdomain conformational shift upon binding the DNA substrate accompanied by local conformational changes in the C-terminal domain zinc finger and N-terminal domain void-filling loop necessary to position the enzyme on the DNA. The detailed biochemical analysis of NEIL2 with an array of oxidized base lesions indicates a significant preference for its lyase activity likely to be paramount when interpreting the biological consequences of variants.

Dynamic and Reversible Decoration of DNA-Based Scaffolds.

An approach to achieving dynamic and reversible decoration of DNA-based scaffolds is demonstrated here. To do this, rationally engineered DNA tiles containing enzyme-responsive strands covalently conjugated to different molecular labels are employed. These strands are designed to be recognized and degraded by specific enzymes (i.e., Ribonuclease H, RNase H, or Uracil DNA Glycosylase, UDG) inducing their spontaneous de-hybridization from the assembled tile and replacement by a new strand conjugated to a different label. Multiple enzyme-responsive strands that specifically respond to different enzymes allow for dynamic, orthogonal, and reversible decoration of the DNA structures. As a proof-of-principle of the strategy, the possibility to orthogonally control the distribution of different labels (i.e., fluorophores and small molecules) on the same scaffold without crosstalk is demonstrated. By doing so, DNA scaffolds that display different antibody recognition patterns are obtained. The approach offers the possibility to control the decoration of higher-order supramolecular assemblies (including origami) with several functional moieties to achieve functional biomaterials with improved adaptability, precision, and sensing capabilities.

Molecular basis of the plant ROS1-mediated active DNA demethylation.

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

Bowhead NEIL1: molecular cloning, characterization, and enzymatic properties.

Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and removes bases, primarily oxidized pyrimidines, which have been damaged by endogenous oxidation or exogenous mutagenic agents. NEIL1 functions through a combined glycosylase/AP (apurinic/apyrimidinic)-lyase activity, whereby it cleaves the N-glycosylic bond between the DNA backbone and the damaged base via its glycosylase activity and hydrolysis of the DNA backbone through beta-delta elimination due to its AP-lyase activity. In our study we investigated our hypothesis proposing that the cancer resistance of the bowhead whale can be associated with a better DNA repair with NEIL1 being upregulated or more active. Here, we report the molecular cloning and characterization of three transcript variants of bowhead whale NEIL1 of which two were homologous to human transcripts. In addition, a novel NEIL1 transcript variant was found. A differential expression of NEIL mRNA was detected in bowhead eye, liver, kidney, and muscle. The A-to-I editing of NEIL1 mRNA was shown to be conserved in the bowhead and two adenosines in the 242Lys codon were subjected to editing. A mass spectroscopy analysis of liver and eye tissue failed to demonstrate the existence of a NEIL1 isoform originating from RNA editing. Recombinant bowhead and human NEIL1 were expressed in E. coli and assayed for enzymatic activity. Both bowhead and human recombinant NEIL1 catalyzed, with similar efficiency, the removal of a 5-hydroxyuracil lesion in a DNA bubble structure. Hence, these results do not support our hypothesis but do not refute the hypothesis either.

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.

Development of QM/MM (ABEEM polarizable force field) method to simulate the excision reaction mechanism of damaged cytosine.

DNA damages are regarded as having harmful effects on cell. The base excision repair mechanism combats these effects by removing damaged bases. The deglycosylation mechanism of excising damaged bases by DNA glycosylase and the state of the leaving base have been controversial. The enzymatic reaction of DNA glycosylase to remove the damaged bases involves not only the formation and breaking of chemical bonds, but also complex polarization effect and charge transfer, which cannot be accurately simulated by the QM/MM method combined with the fixed charge force field. This work has developed the ABEEM fluctuating polarizable force field combining with the QM method, that is (QM/MM[ABEEM]), to accurately simulate the proton transfer, charge transfer and the charge distribution. The piecewise function is used as the valence-state electronegativity in the QM/MM (ABEEM) to realize the accurate fitting of the charge distribution in reaction. And the charge transfer is accurately simulated by the local charge conservation conditions. Four deglycosylation mechanisms including the monofunctional and difunctional mechanisms of four neutral and protonated cytosine derivatives are explored. It is confirmed that the monofunctional mechanism of Asp-activated nucleophile water is a better deglycosylation mechanism and the base is protonated before the reaction occurs. Protonization of the base reduced the activation energy by 10.00-17.00 kcal/mol. Asp provides the necessary charge for the reaction, and DNA glycosylase preferentially cleaves ɛC. This work provides a theoretical basis for the research of excising damaged bases by DNA glycosylase.

DNA Deformation Exerted by Regulatory DNA-Binding Motifs in Human Alkyladenine DNA Glycosylase Promotes Base Flipping.

Human alkyladenine DNA glycosylase (AAG) is a key enzyme that corrects a broad range of alkylated and deaminated nucleobases to maintain genomic integrity. When encountering the lesions, AAG adopts a base-flipping strategy to extrude the target base from the DNA duplex to its active site, thereby cleaving the glycosidic bond. Despite its functional importance, the detailed mechanism of such base extrusion and how AAG distinguishes the lesions from an excess of normal bases both remain elusive. Here, through the Markov state model constructed on extensive all-atom molecular dynamics simulations, we find that the alkylated nucleobase (N3-methyladenine, 3MeA) everts through the DNA major groove. Two key AAG motifs, the intercalation and E131-N146 motifs, play active roles in bending/pressing the DNA backbone and widening the DNA minor groove during 3MeA eversion. In particular, the intercalated residue Y162 is involved in buckling the target site at the early stage of 3MeA eversion. Our traveling-salesman based automated path searching algorithm further revealed that a non-target normal adenine tends to be trapped in an exo site near the active site, which however barely exists for a target base 3MeA. Collectively, these results suggest that the Markov state model combined with traveling-salesman based automated path searching acts as a promising approach for studying complex conformational changes of biomolecules and dissecting the elaborate mechanism of target recognition by this unique enzyme.

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry.

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix-two-turn-helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 - a large insert in its N-terminal domain absent from other DNA glycosylases - is unstructured in solution. It was suggested that helix-two-turn-helix DNA glycosylases undergo open-close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein-protein interactions.

Molecular Mechanisms Associated with Clustered Lesion-Induced Impairment of 8-oxoG Recognition by the Human Glycosylase OGG1.

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, which ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features, and repair have been matters of extensive research; more recently, this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use µs-range molecular dynamics simulations and machine-learning-based postanalysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple-lesion site with a mismatch in 5' or 3'. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5' mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.

Coupling 6-chloro-3-methyluracil with copper: structural features, theoretical analysis, and biofunctional properties.

As nucleobases in RNA and DNA, uracil and 5-methyluracil represent a recognized class of bioactive molecules and versatile ligands for coordination compounds with various biofunctional properties. In this study, 6-chloro-3-methyluracil (Hcmu) was used as an unexplored building block for the self-assembly generation of a new bioactive copper(II) complex, [Cu(cmu)2(H2O)2]·4H2O (1). This compound was isolated as a stable crystalline solid and fully characterized in solution and solid state by a variety of spectroscopic methods (UV-vis, EPR, fluorescence spectroscopy), cyclic voltammetry, X-ray diffraction, and DFT calculations. The structural, topological, H-bonding, and Hirshfeld surface features of 1 were also analyzed in detail. The compound 1 shows a distorted octahedral {CuN2O4} coordination environment with two trans cmu- ligands adopting a bidentate N,O-coordination mode. The monocopper(II) molecular units participate in strong H-bonding interactions with water molecules of crystallization, leading to structural 0D → 3D extension into a 3D H-bonded network with a tfz-d topology. Molecular docking and ADME analysis as well as antibacterial and antioxidant activity studies were performed to assess the bioactivity of 1. In particular, this compound exhibits a prominent antibacterial effect against Gram negative (E. coli, P. aeruginosa) and positive (S. aureus, B. cereus) bacteria. The obtained copper(II) complex also represents the first structurally characterized coordination compound derived from 6-chloro-3-methyluracil, thus introducing this bioactive building block into a family of uracil metal complexes with notable biofunctional properties.

[Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis].

8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity in vitro, that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed KD values ranging from 1.71 to 2.64 µmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.

DNA repair glycosylase hNEIL1 triages damaged bases via competing interaction modes.

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer.

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.

Water-Mediated Oxidation of Guanine by a Repair Enzyme: Simulation Using the ABEEM Polarizable Force Field.

The recognition mechanism of oxidative damage in organisms has long been a research hotspot. Water is an important medium in the recognition process, but its specific role remains unknown. There is a need to develop a suitable force field that can adequately describe the electrostatic, hydrogen bond, and other interactions among the molecules in the complex system of the repair enzyme and oxidized base. The developing ABEEM polarizable force field (PFF) has been used to simulate the repaired enzyme hOGG1 and oxidized DNA (PDB ID: 1EBM) in a biological environment, and the corresponding results are better than those of the fixed-charge force fields OPLS/AA and AMBER OL15. 8-Oxo-G is recognized by Gln315 of hOGG1 mainly through hydrogen bonds mediated by continuous exchange of 2 water molecules. Phe319 and Cys253 are stacked on both sides of the π planes of bases to form sandwich structures. The charge polarization effect gives an important signal to drive the exchange of water molecules and maintains the recognition of oxidation bases by enzymes. The mediated main water molecule A and mediated auxiliary water molecule B together pull Gln315 to recognize 8-oxo-G by hydrogen bond interactions. Then, the charge polarization signal of solvent water molecule C with a large absolute charge causes the absolute charge of O atoms in water molecule A or B to increase by approximately 0.2 e, and water molecule A or B leaves Gln315 and 8-oxo-G. The other water molecule and water molecule C synergistically recognize 8-oxo-G with Gln315. Even though the water molecules between Gln315 and 8-oxo-G are removed, the MD simulation results show that water molecules appear between Gln315 and 8-oxo-G in a very short time (<2 ps). The dwell time of each water molecule is approximately 60 ps. The radial distribution function and dwell time support the correctness of the above mechanism. These polarization effects and hydrogen bonding interactions cannot be simulated by a fixed-charge force field.

DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B1-Induced Formamidopyrimidine Guanine Adduct.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation.

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

Nucleosome Core Particles Lacking H2B or H3 Tails Are Altered Structurally and Have Differential Base Excision Repair Fingerprints.

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (εA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to εA excision by utilizing a global analysis of 21 εA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of εA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.