A Multimodal Approach towards Genomic Identification of Protein Inhibitors of Uracil-DNA Glycosylase.

DNA-mimicking proteins encoded by viruses can modulate processes such as innate cellular immunity. An example is Ung-family uracil-DNA glycosylase inhibition, which prevents Ung-mediated degradation via the stoichiometric protein blockade of the Ung DNA-binding cleft. This is significant where uracil-DNA is a key determinant in the replication and distribution of virus genomes. Unrelated protein folds support a common physicochemical spatial strategy for Ung inhibition, characterised by pronounced sequence plasticity within the diverse fold families. That, and the fact that relatively few template sequences are biochemically verified to encode Ung inhibitor proteins, presents a barrier to the straightforward identification of Ung inhibitors in genomic sequences. In this study, distant homologs of known Ung inhibitors were characterised via structural biology and structure prediction methods. A recombinant cellular survival assay and in vitro biochemical assay were used to screen distant variants and mutants to further explore tolerated sequence plasticity in motifs supporting Ung inhibition. The resulting validated sequence repertoire defines an expanded set of heuristic sequence and biophysical signatures shared by known Ung inhibitor proteins. A computational search of genome database sequences and the results of recombinant tests of selected output sequences obtained are presented here.

dCas9 binding inhibits the initiation of base excision repair in vitro.

Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.

A New Class of Uracil-DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme.

Uracil-DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil-DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 µM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme's active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

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 (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Ablation of SMUG1 Reduces Cell Viability and Increases UVC-Mediated Apoptosis in Hepatocarcinoma HepG2 Cells.

Uracil is an unavoidable aberrant base in DNA sequences, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires multiple biochemical steps with conformational changes of DNA that inhibit DNA replication and interfere with transcription. However, the relevance of uracil in DNA for cellular physiology and transcriptional regulation is not fully understood. We investigated the functional roles of SMUG1 using knock-down (KD) and knock-out (KO) models. The proliferation ratio of SMUG1 KD and KO cells was decreased compared to WT control cells, and the cell cycle was arrested in the G2/M phases before the transition to mitosis. The apoptotic cell death was increased in KD and KO cell lines through the increase of BAX and active caspase 3 expression. Phospho-gamma-H2AX expression, which reflected accumulated DNA damage, was also increased in KO cells. Moreover, the apoptotic cells by DNA damage accumulation were markedly increased in SMUG1 KD and KO cells after ultraviolet C irradiation. Transcriptomic analysis using RNA-seq revealed that SMUG1 was involved in gene sets expression including cell cycle transition and chromatin silencing. Together, the results implicate SMUG1 as a critical factor in cell cycle and transcriptional regulation.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level.

Multiplex DNA methylation and glycosylation are ubiquitous in the human body to ensure the normal function and stability of the genome. The methyltransferases and glycosylases rely on varied enzymes with different action mechanism, which still remain challenges for multiple detection. Herein, we developed a tri-functional dsDNA probe mediated exponential amplification strategy for sensitive detection of human DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG) activities. The tri-functional dsDNA probe was rationally designed with M-DNA and U-DNA. M-DNA contains the 5'-GCmGCGC-3' site for Dnmt1 recognition. U-DNA possesses one uracil as the substrate of UDG and a primer sequence for initiating the amplification reaction. M-DNA was complementary to partial sequence of U-DNA. In the presence of Dnmt1 and UDG, BssHⅡ and Endo Ⅳ were used to nick the 5'-GCGCGC-3' and AP sites respectively, resulting in the release of single-stranded DNA sequence (primer sequence), respectively. After magnetic separation, the released primer sequence hybridizes with padlock DNA (P-DNA), initiating exponential rolling circle amplification to produce numerous G-quadruplexes for recordable signals. The strategy exhibited the limit of detection as low as 0.009 U mL-1 and 0.003 U mL-1 for Dnmt1 and UDG, respectively. Meanwhile, this strategy was successfully applied to detect Dnmt1 and UDG activities in living cell samples at single-cell level and assay the inhibitors of Dnmt1 and UDG. Therefore, the strategy provided a potential method to detect Dnmt1 and UDG activities in biological samples for early clinic diagnosis and therapeutics.

Host-guest recognition coupled with triple signal amplification endows an electrochemiluminescent biosensor with enhanced sensitivity.

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.

Is Uracil-DNA Glycosylase UNG2 a New Cellular Weapon Against HIV-1?

Uracil-DNA glycosylase-2 (UNG2) is a DNA repair protein that removes uracil from single and double-stranded DNA through a basic excision repair process. UNG2 is packaged into new virions by interaction with integrase (IN) and is needed during the early stages of the replication cycle. UNG2 appears to play both a positive and negative role during HIV-1 replication; UNG2 improves the fidelity of reverse transcription but the nuclear isoform of UNG2 participates in the degradation of cDNA and the persistence of the cellular genome by repairing its uracil mismatches. In addition, UNG2 is neutralized by Vpr, which redirects it to the proteasome for degradation, suggesting that UNG2 may be a new cellular restriction factor. So far, we have not understood why HIV-1 imports UNG2 via its IN and why it causes degradation of endogenous UNG2 by redirecting it to the proteasome via Vpr. In this review, we propose to discuss the ambiguous role of UNG2 during the HIV-1 replication cycle.

Target-triggered activation of rolling circle amplification for label-free and sensitive fluorescent uracil-DNA glycosylase activity detection and inhibition.

The expression variations of uracil-DNA glycosylase (UDG) can be used as effective biomarkers for the evaluation of gene regulation and related diseases. Here, by using a new target-triggered activation of rolling circle amplification (RCA) signal enhancement strategy, we have established a sensitive and label-free fluorescent approach for UDG activity detection and inhibition. The target UDG specifically recognizes and excises the uracil bases in a three-strand containing DNA complex to liberate one of the strands. Subsequent ligation of the excised DNA complex converts it into a suitable primer/circular template structure for the initiation of RCA for the generation of long DNA sequences with many repeated G-quadruplexes. Protoporphyrin IX further binds these G-quadruplexes to show substantially enhanced fluorescence to achieve sensitive detecting the activity of UDG with the detection limit as low as 0.00014 U mL-1. Besides, this assay approach has a high specificity toward UDG and can also be utilized to evaluate its inhibition by the uracil glycosylase inhibitor, highlighting the promising applications for convenient and sensitive UDG activity detection and inhibition for disease diagnosis and drug screening.

Targeting uracil-DNA glycosylases for therapeutic outcomes using insights from virus evolution.

Ung-type uracil-DNA glycosylases are frontline defenders of DNA sequence fidelity in bacteria, plants and animals; Ungs also directly assist both innate and humoral immunity. Critically important in viral pathogenesis, whether acting for or against viral DNA persistence, Ungs also have therapeutic relevance to cancer, microbial and parasitic diseases. Ung catalytic specificity is uniquely conserved, yet selective antiviral drugging of the Ung catalytic pocket is tractable. However, more promising precision therapy approaches present themselves via insights from viral strategies, including sequestration or adaptation of Ung for noncanonical roles. A universal Ung inhibition mechanism, converged upon by unrelated viruses, could also inform design of compounds to inhibit specific distinct Ungs. Extrapolating current developments, the character of such novel chemical entities is proposed.

Mass spectrometry-based analysis of macromolecular complexes of Staphylococcus aureus uracil-DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations.

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase (Staphylococcus aureus uracil-DNA glycosylase; SAUDG) and its inhibitor (S. aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these Kd values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins.

Fluorometric determination of the activity of uracil-DNA glycosylase by using graphene oxide and exonuclease I assisted signal amplification.

The base-excision repair enzyme uracil-DNA glycosylase (UDG) plays a crucial role in the maintenance of genome integrity. The authors describe a fluorometric method for the detection of the activity of UDG. It is making use of (a) a 3'-FAM-labeled hairpin DNA probe with two uracil deoxyribonucleotides in the self-complementary duplex region of its hairpin structure, (b) exonuclease I (Exo I) that catalyzes the release of FAM from the UDG-induced stretched ssDNA probe, and (c) graphene oxide that quenches the green FAM fluorescence of the intact hairpin DNA probe in the absence of UDG. If Exo I causes the release of FAM from the hairpin DNA probe, the fluorescence peaking at 517 nm is turned off in the absence of UDG but turned on in its presence. The resulting assay has a wide linear range (0.008 to 1 U·mL-1) and a detection limit as low as 0.005 U·mL-1. It has good specificity for UDG over potentially interfering enzymes and gave satisfactory results when applied to biological samples. Conceivably, the method may be used in a wide range of applications such as in diagnosis, drug screening, and in studying the repair of DNA lesions. Graphical abstract Schematic presentation of a fluorometric strategy for detection of the activity of uracil-DNA glycosylase by using on graphene oxide and exonuclease I assisted signal amplification.

Label-free and high-throughput bioluminescence detection of uracil-DNA glycosylase in cancer cells through tricyclic cascade signal amplification.

We develop a label-free and high-throughput bioluminescence method for the sensitive detection of uracil DNA glycosylase (UDG) through enzyme-mediated tricyclic cascade signal amplification. This method exhibits high sensitivity with a detection limit as low as 0.00031 U mL-1, and it can be further applied for the measurement of enzyme kinetic parameters and the screening of UDG inhibitors as well as cancer cell analysis.

Self-primer and self-template recycle rolling circle amplification strategy for sensitive detection of uracil-DNA glycosylase activity.

Sensitive and accurate detection of uracil-DNA glycosylase (UDG) activity is available for evaluating and validating their function in uracil base-excision repair (UBER) pathway and clinical diagnosis. Here, a sensitive and accurate method for UDG activity detection was developed on the basis of self-primer and self-template recycle rolling circle amplification (Self-RRCA) strategy. First, an immature template (IT) with a uracil base and an Nt.BbvCI nicking site was designed, which could hybridize with a designed primer to form a pre-amplicon probe (PA probe). Under the action of UDG, the uracil base in the PA probe could be removed to generate an apyrimidinic (AP) site. Then the generated AP site was excised by endonuclease IV (endo IV), making the PA probe form a RCA amplicon through reconformation. The RCA amplicon subsequently was used to trigger the RCA, and after Nt.BbvCI nicking reaction, new amplicons were released to initiate next RCA, constituting a Self-RRCA. In this method, the designed IT was not fully complementary with the primer in the ligation part, which could effectively avoid nonspecific ligation reaction and eventually effectively avoid nonspecific amplification. Compared with the linear RCA, the Self-RRCA exhibited higher amplification efficiency. Due to above advantages, a sensitive and accurate detection method was achieved with a limit of 4.68 × 10-5 U mL-1. Furthermore, the method was adopted to screen the inhibitor of UDG and assay the activity of UDG in HeLa cell lysate. This method will offer a promising analysis tool for further biomedical research of UDG and clinical diagnosis.

Integrating DNA structure switch with branched hairpins for the detection of uracil-DNA glycosylase activity and inhibitor screening.

The detection of uracil-DNA glycosylase (UDG) activity is pivotal for its biochemical studies and the development of drugs for UDG-related diseases. Here, we explored an integrated DNA structure switch for high sensitive detection of UDG activity. The DNA structure switch containing two branched hairpins was employed to recognize UDG enzyme and generate fluorescent signal. Under the action of UDG, one branched hairpin was impelled folding into a close conformation after the excision of the single uracil. This reconfigured hairpin could immediately initiate the polymerization/nicking amplification reaction of another branched hairpin accompanying with the release of numerous G-quadruplexes (G4s). In the absence of UDG, the DNA structure switch kept its original configuration, and thus the subsequent polymerization/nicking reaction was inhibited, resulting in the release of few G4 strands. In this work, Thioflavin T was used as signal reporter to target G4s. By integrating the DNA structure switch, the quick response and high sensitivity for UDG determination was achieved and a low detection limit of 0.0001U/mL was obtained, which was superior to the most fluorescent methods for UDG assay. The repeatability of the as-proposed strategy was demonstrated under the concentration of 0.02U/mL and 0.002U/mL, the relative standard deviation obtained from 5 successive samples were 1.7% and 2.8%, respectively. The integrated DNA structure switch strategy proposed here has the potential application for the study of mechanism and function of UDG enzyme and the screening the inhibitors as potential drugs and biochemical tools.

Editing the Genome Without Double-Stranded DNA Breaks.

Genome editing methods have commonly relied on the initial introduction of double-stranded DNA breaks (DSBs), resulting in stochastic insertions, deletions, and translocations at the target genomic locus. To achieve gene correction, these methods typically require the introduction of exogenous DNA repair templates and low-efficiency homologous recombination processes. In this review, we describe alternative, mechanistically motivated strategies to perform chemistry on the genome of unmodified cells without introducing DSBs. One such strategy, base editing, uses chemical and biological insights to directly and permanently convert one target base pair to another. Despite its recent introduction, base editing has already enabled a number of new capabilities and applications in the genome editing community. We summarize these advances here and discuss the new possibilities that this method has unveiled, concluding with a brief analysis of future prospects for genome and transcriptome editing without double-stranded DNA cleavage.

A C-HCR assembly of branched DNA nanostructures for amplified uracil-DNA glycosylase assays.

An autonomous nonenzymatic DNA machine has been successfully engineered based on a two-layered cascaded hybridization chain reaction (C-HCR) circuit, in which the tandem outputs of the upstream HCR-1 unit activate the downstream HCR-2 unit to induce successive repeated hybridizations, generating branched DNA structures and enabling sensitive and selective detection of uracil-DNA glycosylase and its inhibitors.

Chemopreventive Potential of Ethanolic Extracts of Luobuma Leaves (Apocynum venetum L.) in Androgen Insensitive Prostate Cancer.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and ß-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of ß-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.