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Uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG) are the important DNA glycosylases for initiating the repair of DNA damage, and the aberrant expression of DNA glycosylases is closely associated with various diseases, such as Parkinson's disease, several cancers, and human immunodeficiency. The simultaneous detection of UDG and hAAG is helpful for the study of early clinical diagnosis. However, the reported methods for multiple DNA glycosylase assay suffer from the application of an expensive single-molecule instrument, labor-tedious magnetic separation, and complicated design. Herein, we develop a simple fluorescence method with only three necessary DNA strands for the selective and sensitive detection of multiple DNA glycosylase activity based on the generation of 3'-OH terminal-triggered encoding of multicolor fluorescence. The method can achieve the detection limits of 5.5 × 10

DNA nanostructure-based responsive drug delivery has become an increasingly potent method in cancer therapy. However, a variety of important cancer biomarkers have not been explored in searching of new and efficient targeted delivery systems. Uracil degradation glycosylase and human apurinic/apyrimidinic endonuclease are significantly more active in cancer cells. Here, we developed uracil-modified DNA nanotubes that can deliver drugs to tumor cells through an enzyme-induced disassembly process. Although the reaction of these enzymes on their natural DNA substrates has been established, their reactivity on self-assembled nanostructures of nucleic acids is not well understood. We leveraged molecular dynamic simulation based on coarse-grained model to forecast the enzyme reactivity on different DNA designs. The experimental data are highly consistent with the simulation results. It is the first example of molecule simulation being used to guide the design of enzyme-responsive DNA nano-delivery systems. Importantly, we found that the efficiency of drug release from the nanotubes can be regulated by tuning the positions of uracil modification. The DNA nanotubes equipped with cancer-specific aptamer AS1411 are used to deliver doxorubicin to tumor-bearing mice not only effectively inhibiting tumor growth but also protecting major organs from drug-caused damage. We believe that this work provides new knowledge on and insights into future design of enzyme-responsive DNA-based nanocarriers for drug delivery.

Uracil-DNA glycosylase (UDG) initiates base excision repair (BER) by removing damaged or modified nucleobases during DNA repair or mammalian demethylation. The UDG superfamily consists of at least six families with a variety of catalytic specificities and functions. Deinococcus radiodurans, an extreme radiation resistant bacterium, contains multiple members of UDG enzymes within its genome. The present study reveals that the putative protein, DR0022, is a uracil-DNA glycosylase that requires acidic conditions for its glycosylase activity, which is the first case of such an enzyme within the UDG superfamily. The key residues in the catalytic motifs are investigated by biochemical, enzyme kinetics, and de novo structural prediction, as well as molecular modeling analyses. The structural and catalytic roles of several distinct residues are discussed in light of predicted and modeled DR0022 glycosylase structures. The spontaneous mutation rate analysis performed in a dr0022 deficient D. radiodurans strain indicated that the dr0022 gene plays a role in mutation prevention. Furthermore, survival rate analysis in a dr0022 deficient D. radiodurans strain demonstrated its role in stress resistance, including γ-irradiation. Additionally, the novel acid UDG activity in relationship to its in vivo roles is discussed. This work underscores the functional diversity in the UDG superfamily.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10

Uracil-DNA glycosylase (UDG) is a crucial repair enzyme, which is considered a reliable biomarker due to its abnormal expression associated with serious diseases. Herein, DNAzyme-powered cascade walkers were proposed for sensitive detection of UDG. The cascade walkers consisted of a fixed walker and a subsequently activated free walker. The fixed walker was constructed on 13 nm AuNPs by loading a fixed walking strand (WS1) and a track strand 1 (TS1). The WS1 contained a DNAzyme sequence, which was pre-locked by a locking strand (LS) with an uracil base. The TS1 inserted an RNA cleavage site and sealed the same DNAzyme. The free walker tracks were conjugated on 25 nm AuNPs by modifying a FAM-labeled track strand 2 (TS2) with an RNA cleavage site. When UDG specifically recognized the LS, the WS1 was released with the assistance of Endo·IV. Then the WS1 continuously cleaved TS1s to drive the fixed walker, thus releasing many sequences containing DNAzyme. The released sequences acted as free walking strands (WS2s) to repeatedly cleave TS2s to power the free walker, which led to fluorescence accumulation. The cascade walkers successfully detected UDG with a limit of 8.5 × 10

BackgroundOcular infection withToxoplasma gondiiis a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate ofT. gondiiDNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1.MethodsOne hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1.ResultsDetection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg ofT. gondiigenomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection ofToxoplasma gondiiDNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1.ConclusionsData from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Uracil DNA glycosylase is a key enzyme that identifies and removes damaged bases from DNA in the base excision repair pathway. Experimentalists have identified the possibility of Cd(II) reducing the activity of human uracil DNA glycosylase (hUNG) by binding with the enzyme replacing the catalytic water molecule. The present study focus on the stability variation of the enzyme in the presence and absence of Cd(II) and confirms the reported results with the stability analysis done using molecular dynamic (MD) simulation trajectories. The CavityPlus web server identified seven cavities for the free enzyme as possible binding sites and a cavity containing the active site of the enzyme as the best binding cavity for a ligand. Based on the CavityPlus results and the previously reported work, a free hUNG system and two systems of the enzyme with Cd(II); one with Cd(II) replacing the catalytic water molecule in the active site of the enzyme and the other replacing a non-catalytic water molecule in the active site were generated for the simulation. The simulation trajectories were used for the structural stability analysis of the enzyme in all three systems. The binding free energy of the Cd(II) with the enzyme was calculated using molecular mechanics Poisson Boltzmann surface area method. The results showed that the enzyme achieves comparatively high stability with the removal of catalytic water of the enzyme by Cd(II). Therefore, this supports the previously reported idea that Cd(II) replaces catalytic water molecules and affects enzyme activity.

Uracil DNA glycosylase (UDG) is one of the key initiators for the base excision repair pathway. Since abnormal UDG expression is associated with various diseases, sensitive detection of UDG activity is critical for early clinical diagnosis. Here, a smart catalyzed hairpin assembly (CHA)-DNAzyme nanosystem is developed for intracellular UDG imaging by incorporating CHA and DNAzyme onto MnO2 nanosheets. In this strategy, the biodegradable MnO2 nanosheets are employed as nanocarriers for efficiently adsorbing and delivering five DNA probes into cells by endocytosis. Then, the MnO2 nanosheets are degraded by cellular glutathione to release the DNA modules at the same intracellular position. Liberated Mn2+, an indispensable DNAzyme cofactor, was used to promote catalytic cleavage for facilitating the cascade process in cells. Based on the uracil site-recognition and -excision operation of the target UDG, the activated CHA-DNAzyme nanosystem generates lots of DNAzyme-assisted CHA products, turning on the fluorescence resonance energy transfer response. This autocatalytic CHA-DNAzyme nanosystem provides a detectable minimum UDG concentration of 0.23 mU/mL, which is comparable to some reported UDG detection approaches. As a multiple signal amplification strategy, the CHA-DNAzyme nanosystem realizes the UDG imaging in living cells with enhanced sensitivity, indicating great promise in the prediction and diagnosis of early-stage cancer.

The human APOBEC family of eleven cytosine deaminases use RNA and single-stranded DNA (ssDNA) as substrates to deaminate cytosine to uracil. This deamination event has roles in lipid metabolism by altering mRNA coding, adaptive immunity by causing evolution of antibody genes, and innate immunity through inactivation of viral genomes. These benefits come at a cost where some family members, primarily from the APOBEC3 subfamily (APOBEC3A-H, excluding E), can cause off-target deaminations of cytosine to form uracil on transiently single-stranded genomic DNA, which induces mutations that are associated with cancer evolution. Since uracil is only promutagenic, the mutations observed in cancer genomes originate only when uracil is not removed by uracil DNA glycosylase (UNG) or when the UNG-induced abasic site is erroneously repaired. However, when ssDNA is present, replication protein A (RPA) binds and protects the DNA from nucleases or recruits DNA repair proteins, such as UNG. Thus, APOBEC enzymes must compete with RPA to access their substrate. Certain APOBEC enzymes can displace RPA, bind and scan ssDNA efficiently to search for cytosines, and can become highly overexpressed in tumor cells. Depending on the DNA replication conditions and DNA structure, RPA can either be in excess or deficient. Here we discuss the interplay between these factors and how despite RPA, multiple cancer genomes have a mutation bias at cytosines indicative of APOBEC activity.

Here we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand-displacement reactions. The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables the strand displacement reaction. The kinetics of the blocker enzymatic degradation thus controls the time at which the strand displacement reaction starts. By varying the concentration of the blocker strand and the concentration of the enzyme we show that we can finely tune and modulate the delayed onset of the strand displacement reaction. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed strand displacement reactions using RNase H and the two DNA repair enzymes Fpg and UDG and corresponding blockers. The achieved temporal delay can be programmed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show that the delayed strand displacement reactions can be coupled to downstream processes and used to control the onset of ligand release from a DNA nanodevice as well as protein inhibition by a DNA aptamer.

Sensitive monitoring of intracellular uracil-DNA glycosylase (UDG) in living cells is essential to understanding the DNA repair pathways and discovery of anticancer drugs. Herein, we demonstrate the construction of an entropy-driven dumbbell-type DNAzyme assembly circuit for lighting up UDG in living cells via the integration of entropy-driven DNA catalysis (EDC) with the DNAzyme biocatalyst. Target UDG excises the damaged uracil base, causing the breakage of detection probe and the release of trigger. The released trigger can initiate the downstream EDC reaction to form two catalytically active DNAzyme units. The resultant dual Mg

Single-strand selective uracil–DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.

Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD of

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites., C->G transversions can be highly desirable editing outcomes. Here the authors optimise CGBEs and provide a deep learning model for predicting editing outcomes based on sequence context.

Single-nanoparticle inductively coupled plasma mass spectrometry (SP-ICP-MS) has demonstrated unique advantages for the detection of biological samples. However, methods for enzyme activity detection based on SP-ICP-MS technology have been rarely explored. Here we report the development of a novel SP-ICP-MS assay for uracil-DNA glycosylase (UDG) activity detection based on its ability to specifically recognize and remove uracil to induce the cleavage of the DNA probe. Our design allows the generation of single gold nanoparticles correlated to the specific enzymatic reaction for a highly sensitive SP-ICP-MS measurement. The developed assay enables sensitive UDG activity detection with a detection limit of 0.0003 U/mL. The cell lysate analysis by the developed assay reveals its applicability for the detection of UDG activity in real samples. It is envisioned that our design may provide a new paradigm for developing the SP-ICP-MS assay for enzyme activity detection in biological samples.

We demonstrate for the first time the utilization of fluorogenic RNA aptamers for label-free uracil-DNA glycosylase (UDG) assay. Through rationally engineering the transcription machine with dU substitution, this assay requires only a single probe to simultaneously sense and amplify the UDG signal, achieving a low detection limit of 6.3 × 10

5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, diseases including cancer and aging. The demethylation process involves Tet-mediated stepwise oxidation of mC to hmC, fC, or caC, excision of fC or caC by thymine-DNA glycosylase (TDG), and subsequent base excision repair. Thymine-DNA glycosylase (TDG) belongs to uracil-DNA glycosylase (UDG) superfamily, which is a group of enzymes that are initially found to be responsible for excising the deaminated bases from DNA and generating apurinic/apyrimidinic (AP) sites. mC oxidative derivatives may also be generated from Fenton chemistry and γ-irradiation. In screening DNA glycosylase activity in UDG superfamily, we identified new activity on fC- and caC-containing DNA in family 2 MUG/TDG and family 6 HDG enzymes. Surprisingly, we found a glycosylase SMUG2 from bacterium Pedobacter heparinus (Phe), a subfamily of family 3 SMUG1 DNA glycosylase, displayed catalytic activity towards not only DNA containing uracil, but also fC and caC. Given the sequence and structural differences between the family 3 and other family enzymes, we investigated the catalytic mechanism using mutational, enzyme kinetics and molecular modeling approaches. Mutational analysis and kinetics measurements identified I62, N63 and F76 of motif 1, and H205 of motif 2 in Phe SMUG2 as important catalytic residues, of which H205 of motif 2 played a critical role in catalyzing the removal of fC and caC. A catalytic model underlying the roles of these residues was proposed. The structural and catalytic differences between Phe SMUG2 and human TDG were compared by molecular modeling and molecular dynamics simulations. This study expands our understanding of DNA glycosylase capacity in UDG superfamily and provides insights into the molecular mechanism of fC and caC excision in Phe SMUG2.

We demonstrate here the use of DNA repair enzymes to control the assembly of DNA-based structures. To do so, we employed uracil-DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg), two enzymes involved in the base excision repair (BER) pathway. We designed two responsive nucleic acid modules containing mutated bases (deoxyuridine or 8-oxo-7,8-dihydroguanine recognized by UDG and Fpg, respectively) that, upon the enzyme repair activity, release a nucleic acid strand that induces the self-assembly of DNA tiles into tubular structures. The approach is programmable, specific and orthogonal and the two responsive modules can be used in the same solution without crosstalk. This allows to assemble structures formed by two different tiles in which the tile distribution can be accurately predicted as a function of the relative activity of each enzyme. Finally, we show that BER-enzyme inhibitors can also be used to control DNA-tile assembly in a specific and concentration-dependent manner.

Apurinic/apyrimidinic endonuclease 1 (APE1) can selectively incise the AP site of DNA, thus is universal for various DNA substrates for flexible endonuclease-assisted signal amplification. However, the substrate preference of APE1 has never been systematically investigated. Therefore in this work, the detailed sequence-dependent relative activity of APE1 was determined. It turned out that the APE1 activity did vary with the change of the adjacent and opposite bases, and over 10-fold relative activity difference was observed for different sequence combinations. Such difference is appreciable enough to induce evident impact on APE1-involved biosensing. With an APE1 probe designed for cycled signal amplification, the sensitivities followed exactly with the above activity order. Compared with Nb.BbvCl, the sensitivity of the APE1 probe varied between higher and lower than the Nb.BbvCl probe (with varied substrates), demonstrating the importance of the sequence-dependent relative activity of APE1 for optimal biosensor development. Moreover, the above APE1 probe design was harvested and engineered for sensitive biosensing of uracil-DNA glycosylase (UDG). Through theoretical analysis of the interaction between APE1 and the substrates, the accuracy of the determined sequence-dependent relative activity of APE1 was partially confirmed.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (

Highly specific and ultrasensitive detection of uracil-DNA glycosylase (UDG) activity is of great significance for maintaining genomic integrity and medical research of related diseases. Here, we constructed a random DNA walking nanomachine based on a DNAzyme for UDG activity detection on the AuNP (Au nanoparticle) surface. When UDG is present, the U bases in the Y structure are removed, resulting in AP sites, which will be cleaved by Endo-IV to generate a 3' concave end for Exo-III, causing the locking strand of the DNAzyme to be completely hydrolyzed by the Exo-III and release the walking strand to randomly pair with the substrate strand on the AuNP surface; then, the walking strand exerts its cleavage activity with the assistance of Mg2+ to cleave the substrate strand and keep the fluorophore 6-carboxyfluorescein (FAM) away from the surface of the AuNP, which restores the fluorescence signal of this system. In this way, sensitive detection of UDG can be realized, and the detection limit is as low as 3.69 × 10-6 U mL-1. In addition, we found that this method is highly specific to UDG and can be used to detect UDG specifically in complex samples, which has certain application prospects in biomedical research and clinical diagnosis related to UDG.

Background Although CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses. Results As compared to in vitro differentiated MDM, AM from normal donors had sixfold lower levels of dTTP and a sixfold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was eightfold lower in AM compared to the already low levels in MDM. Accordingly, ~ 80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase (DUT) expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors. Conclusions These findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radioisotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the K(m) = 50 nM, V(max) = 0.98 nM/s, and K(cat) = 9.31 s(−1). Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a key role in the persistence of viral infection. We have previously shown that overexpression of an antiviral factor APOBEC3G (A3G) induces hypermutation in duck HBV (DHBV) cccDNA, whereas uracil-DNA-glycosylase (UNG) reduces these mutations. In this study, using cell-culture systems, we examined whether endogenous A3s and UNG affect HBV cccDNA mutation frequency. IFNγ stimulation induced a significant increase in endogenous A3G expression and cccDNA hypermutation. UNG inhibition enhanced the IFNγ-mediated hypermutation frequency. Transfection of reconstructed cccDNA revealed that this enhanced hypermutation caused a reduction in viral replication. These results suggest that the balance of endogenous A3s and UNG activities affects HBV cccDNA mutation and replication competency.

Fatty liver diseases are a major health threat across the western world, leading to cirrhosis and premature morbidity and mortality. Recently, a correlation between the base excision repair enzyme SMUG1 and metabolic homeostasis was identified. As the molecular mechanisms remain unknown, we exploited a SMUG1-knockout mouse model to gain insights into this association by characterizing the liver phenotype in young vs old SMUG1-null mice. We observed increased weight and fat content in one-year old animals, with altered activity of enzymes important for fatty acids influx and uptake. Consistently, lipidomic profiling showed accumulation of free fatty acids and triglycerides in SMUG1-null livers. Old SMUG1-knockout mice also displayed increased hepatocyte senescence and DNA damage at telomeres. Interestingly, RNA sequencing revealed widespread changes in the expression of lipid metabolic genes already in three months old animals. In summary, SMUG1 modulates fat metabolism favouring net lipogenesis and resulting in development of a fatty liver phenotype.

Uracil-DNA glycosylase (UDG) is essential to the maintenance of genomic integrity due to its critical role in base excision repair pathway. However, existing UDG assays suffer from laborious procedures, poor specificity, and limited sensitivity. In this research, we construct a catalytic single-molecule Föster resonance energy transfer (FRET) biosensor for in vitro and in vivo biosensing of UDG activity. Target UDG can remove uracil base from the detection probe and cause the cleavage of detection probe by apurinic/apyrimidinic endonuclease (APE1), which exposes its toehold domain and initiates catalytic assembly of two fluorescently labeled hairpin probes via toehold-meditated strand displacement reaction (SDA) to generate abundant DNA duplexes with amplified FRET signal. In this assay, target UDG signal is amplified via enzyme-free catalytic reaction and the whole reaction may be completed in one step, which greatly simplifies the assay procedure, reduces the assay time, and facilitates the cellular imaging. This biosensor enables specific and sensitive measurement of UDG down to 0.00029 U/mL, and it is suitable for analyzing kinetic parameters, screening inhibitors, and even imaging endogenous UDG in live cells. Importantly, this biosensor can visually quantify various DNA repair enzymes by rationally altering DNA substrates.

Cancer therapeutics produce reactive oxygen species (ROS) that damage the cancer genome and lead to cell death. However, cancer cells can resist ROS-induced cytotoxicity and survive. We show that nuclear-localized uracil-DNA N-glycosylase isoform 2 (UNG2) has a critical role in preventing ROS-induced DNA damage and enabling cancer-cell resistance. Under physiological conditions, UNG2 is targeted for rapid degradation via an interaction with the E3 ligase UHRF1. In response to ROS, however, UNG2 protein in cancer cells exhibits a remarkably extended half-life. Upon ROS exposure, UNG2 is deacetylated at lysine 78 by histone deacetylases, which prevents the UNG2-UHRF1 interaction. Accumulated UNG2 protein can thus excise the base damaged by ROS and enable the cell to survive these otherwise toxic conditions. Consequently, combining HDAC inhibitors (to permit UNG2 degradation) with genotoxic agents (to produce cytotoxic cellular levels of ROS) leads to a robust synergistic killing effect in cancer cells in vitro. Altogether, these data support the application of a novel approach to cancer treatment based on promoting UNG2 degradation by altering its acetylation status using an HDAC inhibitor.

Uracil-DNA glycosylases (UDGs) are conserved DNA-repair enzymes that can be found in many species, including herpesviruses. Since they play crucial roles for efficient viral DNA replication in herpesviruses, they have been considered as potential antiviral targets. In our previous work, Staphylococcus aureus SAUGI was identified as a DNA mimic protein that targets UDGs from S. aureus, human, Herpes simplex virus (HSV) and Epstein-Barr virus (EBV). Interestingly, SAUGI has the strongest inhibitory effects with EBVUDG. Here, we determined complex structures of SAUGI with EBVUDG and another γ-herpesvirus UDG from Kaposi's sarcoma-associated herpesvirus (KSHVUDG), which SAUGI fails to effectively inhibit. Structural analysis of the SAUGI/EBVUDG complex suggests that the additional interaction between SAUGI and the leucine loop may explain why SAUGI shows the highest binding capacity with EBVUDG. In contrast, SAUGI appears to make only partial contacts with the key components responsible for the compression and stabilization of the DNA backbone in the leucine loop extension of KSHVUDG. The findings in this study provide a molecular explanation for the differential inhibitory effects and binding strengths that SAUGI has on these two UDGs, and the structural basis of the differences should be helpful in developing inhibitors that would interfere with viral DNA replication.

Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions., REV7 has emerged as a critical regulator of DNA double-strand breaks repair. Here, the authors show that REV7 is crucial for both antibody class switch recombination and somatic hypermutation in activated B cells, in addition to their survival upon AID-deamination.

Abstract Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

A novel fluorescence sensing strategy for ultrasensitive and highly specific detection of adenosine triphosphate (ATP) has been developed by the combination of the proximity ligation assay with bidirectional enzymatic repairing amplification (BERA). The strategy relies on proximity binding-triggered the release of palindromic tail that initiates bidirectional cyclic enzymatic repairing amplification reaction with the aid of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). A fluorescence-quenched hairpin probe with a palindromic tail at the 3' end is skillfully designed that functions as not only the recognition element, primer, and polymerization template for BERA but also the indicator for fluorescence signal output. On the basis of the amplification strategy, this biosensor displays excellent sensitivity and selectivity for ATP detection with an outstanding detection limit of 0.81 pM. Through simultaneously enhancing the target response signal value and reducing nonspecific background, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, our biosensor also shows promising potential in real sample analysis. Therefore, the proximity-enabled BERA strategy indeed creates a simple and valuable fluorescence sensing platform for ATP identification and related disease diagnosis and biomedical research.

A novel genotoxicity sensor was developed based on the base repair process associated with the electrochemiluminescence (ECL) detection of abasic sites in a double-stranded DNA monolayer. This is the first time that an ECL sensor with the ability to identify the removed nucleobases in a DNA duplex has been studied. The successful detection of abasic sites created by DNA glycosylase indicates its further applications for examining some other specific types of DNA damage.

Loop-mediated isothermal amplification (LAMP) is a useful molecular biology technology for analytical applications, but it is prone to contamination because of escaped aerosols, leading to false positive results. This report establishes an integrated, rapid, and accurate method to detect bacteria in urine samples by incorporating uracil-DNA-glycosylase (UDG) into real-time loop-mediated isothermal amplification (RT-LAMP). To do this, nucleic acids from five clinically important uropathogens, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Enterococcus faecalis, were directly captured and concentrated using Flinders Technology Associates (FTA) Elute cards. Following elution, the extracted DNAs were specifically amplified with sets of LAMP primers. The added UDG in the modified LAMP reaction excises uracils from previously amplified products or contaminants and generates apyrimidinic (AP) sites, reducing false positive rates. This UDG-assisted RT LAMP strategy was able to degrade carryover contaminants to as little as 1 femtogram (10-15 g). The assay showed a limit of detection of 104 CFU mL-1 with a sensitivity of 94.1% and a specificity of 95.0%. Both the sensitivity and specificity were improved compared to LAMP carried out without UDG. Our results indicate that the UDG-assisted RT LAMP is of great potential for rapid and precise analysis of nucleic acids in real applications.

We demonstrate for the first time that host-guest recognition coupled with triple signal amplification endows an electrochemiluminescent (ECL) biosensor with enhanced sensitivity for uracil DNA glycosylase (UDG) assay. This biosensor exhibits good selectivity and extremely high sensitivity, and it can be used to screen UDG inhibitors and measure the cellular UDG activity as well.

As one of the initiating DNA glycosylases in the base excision repair pathway, Uracil-DNA glycosylase (UDG) plays a pivotal role in maintaining genomic integrity. The abnormal expression of UDG in the organism is highly relevant to multiple diseases. Thus, rapid and sensitive detection of UDG activity is essential to aid early clinical diagnosis and biomedical research. Here we developed a rapid, sensitive and selective biosensor for UDG activity detection based on the substrate preference of Lambda exonuclease (λ exo). The protruding end in the substrate produced by UDG could be digested at a markedly high rate by λ exo, generating a detectable fluorescence signal. This proposed strategy for UDG detection exhibited high selectivity and high sensitivity (0.0001 U/mL) in a short time. It has also been successfully applied to detect UDG in real biological samples and the screening of UDG inhibitors.