DNA Glycosylase Activity Research Articles

Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase.

We herein describe A novel strategy to accurately determine uracil DNA glycosylase (UDG) activity is described based on the finding that nicking endonuclease-assisted cleavage reaction can be regulated by the presence of abasic site. This strategy utilizes DNA probes rationally designed to contain uracil base at the cleavage site for nicking endonuclease, which is coupled to the isothermal nicking endonuclease amplification reaction (NEAR) method. In the absence of UDG, intact DNA probes generate a large number of double-stranded (ds) DNA products through the NEAR, but the presence of UDG that converts uracil base into abasic site suppresses nicking endonuclease activity and the subsequent NEAR. As a result, dsDNA products are not produced, which is simply monitored by the dsDNA specific fluorescence dye, SYBR green I. By employing this strategy, we sensitively determined the UDG activity down to 0.003 U mL-1 with high specificity over other base excision enzymes. In addition, the diagnostic capability of this method was successfully verified by reliably assaying UDG present in a human serum sample.

DNA three way junction-mediated recycling amplification for sensitive electrochemical monitoring of uracil-DNA glycosylase activity and inhibition

Abstract The monitoring of uracil DNA glycosylase (UDG) activity in a simple and sensitive way is very important to clinical diagnosis and biochemical research. In this paper, based on a DNA three junction-mediated recycling signal amplification strategy, we have developed a sensitive and convenient electrochemical approach for monitoring UDG activity and its inhibition. The target UDG specifically recognizes and hydrolyzes uracil bases located in the DNA duplexes, causing the release of the methylene blue-tagged ssDNAs (MB-DNAs) to trigger the disassembly of the three way junction (TWJ) probes and the attachment of the MB-DNAs on the sensor electrode through toehold-mediated strand displacement reaction (TSDR). The disassembled sequences from the TWJ probes further initiate another TSDR to cause cyclic capture of lots of MB-DNAs on the sensing electrode, generating significantly amplified current outputs for sensitively detecting UDG at the low 0.00028 U/mL level. Moreover, such UDG sensing method shows high sensitivity and can be used to screen the inhibition effect of the uracil glycosylase inhibitor to UDG activity, demonstrating the great potential of the developed sensing method for clinical applications.

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation.

Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer.

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297)

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8μM-92.7μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

Identification of a prototypical single-stranded uracil DNA glycosylase from Listeria innocua

A recent phylogenetic study on UDG superfamily estimated a new clade of family 3 enzymes (SMUG1-like), which shares a lower homology with canonic SMUG1 enzymes. The enzymatic properties of the newly found putative DNA glycosylase are unknown. To test the potential UDG activity and evaluate phylogenetic classification, we isolated one SMUG1-like glycosylase representative from Listeria innocua (Lin). A biochemical screening of DNA glycosylase activity in vitro indicates that Lin SMUG1-like glycosylase is a single-strand selective uracil DNA glycosylase. The UDG activity on DNA bubble structures provides clue to its physiological significance in vivo. Mutagenesis and molecular modeling analyses reveal that Lin SMUG1-like glycosylase has similar functional motifs with SMUG1 enzymes; however, it contains a distinct catalytic doublet S67-S68 in motif 1 that is not found in any families in the UDG superfamily. Experimental investigation shows that the S67M-S68N double mutant is catalytically more active than either S67M or S68N single mutant. Coupled with mutual information analysis, the results indicate a high degree of correlation in the evolution of SMUG1-like enzymes. This study underscores the functional and catalytic diversity in the evolution of enzymes in UDG superfamily.

Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity

We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications.

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

Uracil DNA glycosylase (UDG) activities in Bradyrhizobium diazoefficiens: characterization of a new class of UDG with broad substrate specificity.

Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.

Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated invivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT2 -mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

Mutation Spectrum Induced by 8-Bromoguanine, a Base Damaged by Reactive Brominating Species, in Human Cells.

To date, the types of mutations caused by 8-bromoguanine (8BrG), a major base lesion induced by reactive brominating species during inflammation, in human cells and the 8BrG repair system remain largely unknown. In this study, we performed a supF forward mutation assay using a shuttle vector plasmid containing a single 8BrG in three kinds of human cell lines and revealed that 8BrG in DNA predominantly induces a G → T mutation but can also induce G → C, G → A, and delG mutations in human cells. Next, we tested whether eight kinds of DNA glycosylases (MUTYH, MPG, NEIL1, OGG1, SMUG1, TDG, UNG2, and NTHL1) are capable of repairing 8BrG mispairs with any of the four bases using a DNA cleavage activity assay. We found that both the SMUG1 protein and the TDG protein exhibit DNA glycosylase activity against thymine mispaired with 8BrG and that the MUTYH protein exhibits DNA glycosylase activity against adenine mispaired with 8BrG. These results suggest that 8BrG induces some types of mutations, chiefly a G → T mutation, in human cells, and some DNA glycosylases are involved in the repair of 8BrG.

Cyclic enzymatic repairing-mediated dual-signal amplification for real-time monitoring of thymine DNA glycosylase.

We develop a new fluorescence method for real-time monitoring of thymine DNA glycosylase (TDG) activity through cyclic enzymatic repairing-mediated dual-signal amplification. This method exhibits excellent sensitivity with a detection limit of 5.6 × 10-7 U μL-1, and it can be used to determine kinetic parameters and quantify TDG activity from even single cancer cells.

Restriction glycosylases: involvement of endonuclease activities in the restriction process.

All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction–modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses.

ABSTRACTThe temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity.IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity.

Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation

We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research.

A G-quadruplex-selective luminescent probe with an anchor tail for the switch-on detection of thymine DNA glycosylase activity

Thymine DNA glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation, which also plays an essential role in DNA demethylation. In this work, the novel iridium(III) complex 1 with an anchor tail was synthesized and employed to construct a G-quadruplex-based assay for detecting TDG activity in aqueous solution by using the mismatched base excising property of TDG with T4 DNA ligase and phi29 DNA polymerase, in concert with the rolling circle amplification (RCA) strategy. The assay achieved a detection limit of 0.048UmL−1 (0.012ngmL−1), and showed high selectivity towards TDG even in the presence of other proteins and enzymes. Additionally, the assay could function in diluted cellular debris.

Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.

DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase.

Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing.

Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring.

AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair.

Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3'-5' exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3'-5' exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER.

Functional Evaluation of Nine Missense-Type Variants of the Human DNA Glycosylase Enzyme MUTYH in the Japanese Population.

Biallelic germline mutations of MUTYH, the gene encoding DNA glycosylase, cause MUTYH-associated polyposis (MAP), characterized by multiple colorectal adenomas and carcinoma(s). However, a considerable number of MUTYH variants are still functionally uncharacterized. Herein, we report the results of functional evaluation of nine missense-type MUTYH variant proteins in the Japanese population. The DNA glycosylase activity and ability to suppress mutations caused by 8-hydroxyguanine, an oxidized form of guanine, were examined for the nine variants of type 2 MUTYH, a nuclear form of the enzyme, by DNA cleavage activity assay and supF forward mutation assay, respectively. Both activities were severely defective in the p.N210S MUTYH type 2 variant corresponding to p.N238S in the reference MUTYH form and partially defective in p.R219G variant corresponding to p.R247G, but nearly fully retained in seven other variants examined. Our results suggest that p.N238S and p.R247G are likely to be pathogenic alleles for MAP.