Human Uracil DNA Glycosylase Research Articles

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

Computational investigation of impact of Pb(II) and Ni(II) ions on hUNG enzyme: insights from molecular dynamics simulations

Human uracil DNA glycosylase (hUNG), a crucial player in the initiation of the base excision repair pathway, is susceptible to alterations in function and conformation induced by the accumulation of toxic metals. Despite the recognized impact of toxic metals on DNA repair enzymes, there exists a notable deficiency in theoretical investigations addressing this phenomenon. This study investigates the impact of toxic heavy metal ions, Pb(II) and Ni(II), on the stability of hUNG through molecular dynamics (MD) simulations. The initial analysis involved the identification of key cavities in the hUNG enzyme. Notably, the active site cavity emerged as a promising site for ligand binding. Subsequently, AutoDockTools software was employed to dock Pb(II) and Ni(II) onto the identified cavities, followed by extensive MD simulations. The MD analysis, encompassing parameters such as root mean square deviation, radius of gyration, solvent accessible surface area, hydrogen bond variations, Ramachandran plot, principal component analysis, and root mean square fluctuations, collectively revealed distinct alterations in the behavior of the enzyme upon complexation with Pb(II) and Ni(II). Interestingly, the enzyme exhibited enhanced structural stability, reduced flexibility, and modified hydrogen bonding patterns in the presence of these toxic metal ions. The observed limitation in structural flexibility implies a more rigid and stable conformation when the enzyme complex with Pb(II) and Ni(II) compared to its free form. This structural alteration may lead to a potential reduction in enzymatic activity, suggesting that toxic metal ions influence the functional dynamics of hUNG. These computational findings offer valuable insights into the molecular interactions between metal ions and enzymes. Communicated by Ramaswamy H. Sarma

Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation.

DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA-dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins.

Assay design for analysis of human uracil DNA glycosylase.

Human uracil DNA glycosylase (UNG2) is an enzyme whose primary function is to remove uracil bases from genomic DNA. UNG2 activity is critical when uracil bases are elevated in DNA during class switch recombination and somatic hypermutation, and additionally, UNG2 affects the efficacy of thymidylate synthase inhibitors that increase genomic uracil levels. Here, we summarize the enzymatic properties of UNG2 and its mitochondrial analog UNG1. To facilitate studies on the activity of these highly conserved proteins, we discuss three fluorescence-based enzyme assays that have informed much of our understanding on UNG2 function. The assays use synthetic DNA oligonucleotide substrates with uracil bases incorporated in the DNA, and the substrates can be single-stranded, double-stranded, or form other structures such as DNA hairpins or junctions. The fluorescence signal reporting uracil base excision by UNG2 is detected in different ways: (1) Excision of uracil from end-labeled oligonucleotides is measured by visualizing UNG2 reaction products with denaturing PAGE; (2) Uracil excision from dsDNA substrates is detected in solution by base pairing uracil with 2-aminopurine, whose intrinsic fluorescence is enhanced upon uracil excision; or (3) UNG2 excision of uracil from a hairpin molecular beacon substrate changes the structure of the substrate and turns on fluorescence by relieving a fluorescence quench. In addition to their utility in characterizing UNG2 properties, these assays are being adapted to discover inhibitors of the enzyme and to determine how protein-protein interactions affect UNG2 function.

Effect of Uracil DNA Glycosylase Activity on the Efficacy of Thymidylate Synthase Inhibitor/HDAC Inhibitor Combination Therapies in Colon Cancer

Human uracil DNA glycosylase (UNG2) is responsible for removal of uracil bases from DNA and initiates base excision repair pathways. Accumulation of uracil or its fluorinated analogs in DNA is one of the killing mechanisms of thymidylate synthase (TS) inhibitors in cancer cells, and depletion of UNG2 often enhances the toxicity of these anticancer drugs. We used CRISPR to knockout UNG2 from HT29 colon cancer cells and confirmed the absence of protein by western blot and uracil excision assays. We tested the effect of UNG2 KO on the efficacy of multiple TS inhibitors (5‐fluorouracil, fluorodeoxyuridine, pemetrexed, and raltitrexed), and we determined that only fluorodeoxyuridine and raltitrexed were significantly more potent in UNG2 KO cells compared to wild‐type HT29 cells (fluorodeoxyuridine IC50: 2 mM (wt) vs. 3 nM (KO); raltitrexed IC50: 14 nM (wt) vs. 2 nM (KO)). Interestingly, UNG2 protein levels can also be depleted by the HDAC inhibitors SAHA and MS275, providing a pharmacologic strategy to reduce UNG2 activity in cells. Unexpectedly, the HDAC inhibitors synergized with 5‐fluorouracil, but not fluorodeoxyuridine, in both wild‐type and UNG2‐knockout cells. This suggested that HDAC inhibitors sensitized cells to 5‐fluorouracil through an UNG2‐independent mechanism. Moreover, cell death pathways activated by fluorodeoxyuridine and regulated by UNG2 activity are not sensitized by HDAC inhibitors. Our combined genetic and pharmacologic strategies targeting UNG2 activity in cells are defining cell death mechanisms for combination therapies of TS inhibitors and HDAC inhibitors. Future work will examine these drug combinations in additional cell lines to understand optimal therapeutic combinations and to further refine mechanisms of cell death.

Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification.

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

Impact of Cd(II) on the stability of human uracil DNA glycosylase enzyme; an implication of molecular dynamics trajectories on stability analysis

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.

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.

An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation

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 <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and “door stopper” strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.

Abstract 1390: Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing

Abstract Previously reported shRNA knockdown studies have established that expression of the DNA repair enzyme human uracil DNA glycosylase (hUNG2) is protective against 5-flourodeoxyuridine (5FdUrd) cell killing in p53 mutated cancer cell lines, but not in cell lines with functional p53. Here we take a different approach to understanding the role of hUNG2 in 5FdUrd chemotherapy by selectively inhibiting the catalytic activity of hUNG2 using the small bacterial inhibitor protein UGI. UGI binds extremely tightly to the DNA binding and catalytic site of hUNG2, effectively mimicking the impact of a small molecule inhibitor and allowing us to explore the potential therapeutic vulnerabilities that loss of hUNG2 activity produces in p53 mutated and wild-type cancer cells. Accordingly, we used lentiviral transduction to introduce UGI, under control of a doxycycline-inducible promoter, into a large panel of cancer cell lines. In vitro hUNG2 activity measurements using cell extracts established that all lines had robust activity before induction of UGI and undetectable activity after induction. We find that some wild-type and mutant p53 cancer lines from different tissues show significant increases in drug sensitivity. For instance, the p53 wild-type MCF7 breast cancer line showed a 10,000-fold increase in sensitivity, while the p53 mutant HT29 colorectal cancer line showed a 50-fold increased sensitivity. Overall, the results do not support the proposal that p53 status itself determines whether a cancer cell will be sensitized to 5FdUrd in the presence of hUNG2 inhibition. Ongoing studies are focused on identifying the cellular factors that contribute to drug sensitivity under conditions of hUNG2 inhibition. Realizing the full therapeutic benefit of 5FdUrd requires identification of the cancer cell phenotypes that are sensitized upon hUNG2 inhibition. Citation Format: Eric S. Christenson, Junru Cui, Anthony Gizzi, Mesfin Meshesha, James T. Stivers. Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1390.

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages.

Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription (“uracilation”). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.

Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1

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.

Macromolecular crowding induces compaction and DNA binding in the disordered N-terminal domain of hUNG2

Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.

Mechanism of Facilitated Diffusion of DNA Repair Proteins in Crowded Environment: Case Study with Human Uracil DNA Glycosylase.

Preserving the genomic integrity is a fundamental requirement, primarily achieved by the DNA repair proteins through their continuous patrolling on the DNA in search of lesions. Human uracil DNA glycosylase (hUNG) is one such DNA repair protein that recognizes uracil in the duplex DNA and excises it using the extrahelical base recognition mechanism. Recent site transfer assay experiments based on full-length hUNG suggest that a crowded environment facilitates its search efficiency, which is enhanced further in the presence of a 93 residue disordered tail associated with its N-terminal. In this study, by performing extensive molecular dynamics simulations with an appropriately tuned model of protein and DNA in the presence of inert crowding agents, we probe the role of cellular crowding and the disordered region in the target search efficiency of the enzyme. Our analysis highlights a complex interplay among the shape of the enzyme, the presence of a disordered tail, and the macromolecular crowding agents that work in harmony to enhance the facilitated diffusion of hUNG protein in a crowded environment. The findings provide novel insights into the in vivo target search mechanism of DNA repair proteins.

The Role of Active-Site Residues Phe98, His239, and Arg243 in DNA Binding and in the Catalysis of Human Uracil-DNA Glycosylase SMUG1.

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme–DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme–DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.

Abstract 1663: Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies

Abstract Our research aims to develop therapeutically active inhibitors of the human uracil-DNA glycosylase (UNG). UNG, a highly conserved enzyme, plays a pivotal role in preventing abnormal bases, uracil and 5-fluorouracil, from incorporation into DNA as a result of treatment with chemotherapeutic drugs, floxuridine (5-FdU) and pemetrexed (PEM). Our previous publication reports that the depletion of UNG by shRNA causes uracil and 5-FU incorporation into DNA following 5-FdU or PEM exposure, and highly sensitizes certain human cancer cells to these chemotherapeutic drugs. These data suggest that UNG specific inhibitors could improve the anticancer effect of 5-FdU or PEM. However, a few known inhibitors have not been shown to function at therapeutic concentrations in human cells. Therefore, in order to identify a novel, small-molecule compound targeting the human UNG enzymatic activity, we have previously optimized a biochemically high-throughput screening (HTS) assay that measures the removal of uracil from a DNA hairpin based on the un-quenching of fluorescence. This assay has been successfully employed in a pharmacologically active compound collection screen (&amp;gt;3,000 small-molecules+ approved drugs). In 384-well plate runs using control wells in which enzyme is not added, the average assay provides signal to background ratios of 3.0 to 4.0. Typical Z factors range from 0.6-0.7, indicating a robust assay. We have identified five bioactive compounds with IC50 values &amp;lt; 1uM and have selected a nanoMolar inhibitor of the human UNG enzyme termed, UNGi-A. By using an in vitro DNA glycosylase activity assay, this compound displays a significant inhibitory activity towards the purified human UNG enzyme over time, in a dose-dependent manner. Further characterization shows a significant effect of this inhibitor on UNG activity in cellular extract that expresses endogenous UNG from DLD1 human colon cancer cell line. The result suggests that UNGi-A compound diminishes UNG activity in cell extracts. In addition, thermal shift assay (TSA) data suggest that UNGi-A binds directly to the human UNG enzyme. Together, this compound shows potential activity that blocks the human UNG activity and we are currently evaluating the effect of UNGi-A in combination with 5-FdU on UNG activity in human cancer cells. Citation Format: Mya T. Nguyen, Yan Yan, Yuriy Fedorvo, John Pink, Drew Adams, Stanton Gerson. Identification and characterization of a small-molecule inhibitor of the human uracil-DNA glycosylase for use in combination with DNA damage-based anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1663.

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions.

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA–dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA–dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for uracil located <10 bp from the junction, but only when the overhang had a 5′ end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA–dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of uracils that arise from dUTP incorporation during DNA replication, or additionally, uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination.

Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA.

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.

Characterizing the Enhanced Nanoscale Translocation Properties of hUNG2 Facilitated by its Disordered N-terminal Domain In Vitro and in Human Cells

Nuclear localized human uracil DNA glycosylase 2 (hUNG2) initiates base excision repair (BER) of uracil lesions in genomic DNA. Similar to many DNA glycosylases, hUNG2 possesses a ∼90 amino acid unstructured N-terminal domain (NTD) that encodes the nuclear localization signal as well as several sites for post translational modification and interaction with replication proteins. Here, we present evidence that within crowded environments such as the cell nucleus, the hUNG2 NTD endows enhanced nanoscale translocation properties to the enzyme. Based on in vitro measurements comparing hUNG2 to a catalytic hUNG2 construct lacking the NTD under crowded conditions, these enhancements are manifested in a near 10-fold DNA length dependent increase in kcat, a ∼3-fold increase in the overall catalytic efficiency (kcat/KM), and an improved ability for the enzyme to translocate between damaged sites separated greater than 20 bp. These translocation studies were further implemented in human cells to develop the first in vivo assay of its kind, able to detect nanoscale translocation events of hUNG2 between uracil lesion sites 10 to 80 bp apart. hUNG2 translocation between target sites in human cells increased over that seen under similar conditions in vitro. These findings together reveal a novel role for both the hUNG2 NTD as well as the crowded nuclear environment in facilitating DNA damage detection.