Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

Test strip coupled Cas12a-assisted signal amplification strategy for sensitive detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a base excision repair (BER) enzyme, which catalyzes the hydrolysis of uracil bases in DNA chains that contain uracil and N-glycosidic bonds of the sugar phosphate backbone. The expression of UDG enzyme is associated with a variety of genetic diseases including cancers. Hence, the identification of UDG activity in cellular processes holds immense importance for clinical investigation and diagnosis. In this study, we employed Cas12a protein and enzyme-assisted cycle amplification technology with a test strip to establish a precise platform for the detection of UDG enzyme. The designed platform enabled amplifying and releasing the target probe by reacting with the UDG enzyme. The amplified target probe can subsequently fuse with crRNA and Cas12a protein, stimulating the activation of the Cas12a protein to cleave the signal probe, ultimately generating a fluorescent signal. This technique showed the ability for evaluating UDG enzyme activity in different cell lysates. In addition, we have designed a detection probe to convert the fluorescence signal into test strip bands that can then be observed with the naked eye. Hence, our tool presented potential in both biomedical research and clinical diagnosis related to DNA repair enzymes.

Structural and functional coupling in cross-linking uracil-DNA glycosylase UDGX.

Enzymes in uracil-DNA glycosylase (UDG) superfamily are involved in removal of deaminated nucleobases such as uracil, methylcytosine derivatives such as formylcytosine and carboxylcytosine, and other base damage in DNA repair. UDGX is the latest addition of a new class to the UDG superfamily with a sporadic distribution in bacteria. UDGX type enzymes have a distinct biochemical property of cross-linking itself to the resulting AP site after uracil removal. Built on previous biochemical and structural analyses, this work comprehensively investigated the kinetic and enzymatic properties of Mycobacterium smegmatis UDGX. Kinetics and mutational analyses, coupled with structural information, defined the roles of E52, D56, D59, F65 of motif 1, H178 of motif 2 and N91, K94, R107 and H109 of motif 3 play in uracil excision and cross-linking. More importantly, a series of quantitative analyses underscored the structural coupling through inter-motif and intra-motif interactions and subsequent functional coupling of the uracil excision and cross-linking reactions. A catalytic model is proposed, which underlies this catalytic feature unique to UDGX type enzymes. This study offers new insight on the catalytic mechanism of UDGX and provides a unique example of enzyme evolution.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

Divergent structures of Mammalian and gammaherpesvirus uracil DNA glycosylases confer distinct DNA binding and substrate activity.

Uracil DNA glycosylase (UNG) removes mutagenic uracil base from DNA to initiate base excision repair (BER). The result is an abasic site (AP site) that is further processed by the high-fidelity BER pathway to complete repair and maintain genome integrity. The gammaherpesviruses (GHVs), human Kaposi sarcoma herpesvirus (KSHV), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68) encode functional UNGs that have a role in viral genome replication. Mammalian and GHVs UNG share overall structure and sequence similarity except for a divergent amino-terminal domain and a leucine loop motif in the DNA binding domain that varies in sequence and length. To determine if divergent domains contribute to functional differences between GHV and mammalian UNGs, we analyzed their roles in DNA interaction and catalysis. By utilizing chimeric UNGs with swapped domains we found that the leucine loop in GHV, but not mammalian UNGs facilitates interaction with AP sites and that the amino-terminal domain modulates this interaction. We also found that the leucine loop structure contributes to differential UDGase activity on uracil in single- versus double-stranded DNA. Taken together we demonstrate that the GHV UNGs evolved divergent domains from their mammalian counterparts that contribute to differential biochemical properties from their mammalian counterparts.

Correlated Target Search by Vaccinia Virus Uracil-DNA Glycosylase, a DNA Repair Enzyme and a Processivity Factor of Viral Replication Machinery.

The protein encoded by the vaccinia virus D4R gene has base excision repair uracil-DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

Rolling circle transcription and CRISPR/Cas12a-assisted versatile bicyclic cascade amplification assay for sensitive uracil-DNA glycosylase detection.

Uracil-DNA glycosylase (UDG) is pivotal in maintaining genome integrity and aberrant expressed UDG is highly relevant to numerous diseases. Sensitive and accurate detecting UDG is critically significant for early clinical diagnosis. In this research, we demonstrated a sensitive UDG fluorescent assay based on rolling circle transcription (RCT)/CRISPR/Cas12a-assisted bicyclic cascade amplification strategy. Target UDG catalyzed to remove uracil base of DNA dumbbell-shape substrate probe (SubUDG) to produce an apurinic/apyrimidinic (AP) site, at which SubUDG was cleaved by apurinic/apyrimidinic endonuclease (APE1) subsequently. The exposed 5'-PO4 was ligated with the free 3'-OH terminus to form an enclosed DNA dumbbell-shape substrate probe (E-SubUDG). E-SubUDG functioned as a template can actuate T7 RNA polymerase-mediated RCT signal amplification, generating multitudes of crRNA repeats. The resultant Cas12a/crRNA/activator ternary complex activated the activity of Cas12a, causing a significantly enhanced fluorescence output. In this bicyclic cascade strategy, target UDG was amplified via RCT and CRISPR/Cas12a, and the whole reaction was completed without complex procedures. This method enabled sensitive and specific monitor UDG down to 0.0005 U/mL, screen corresponding inhibitors, and analyze endogenous UDG in A549 cells at single-cell level. Importantly, this assay can be extended to analyze other DNA glycosylase (hAAG and Fpg) by altering the recognition site in DNA substrates probe rationally, thereby offering a potent tool for DNA glycosylase-associated clinical diagnosis and biomedical research.

Ionic strength modulates excision of uracil by SMUG1 from nucleosome core particles.

Ionic strength affects many cellular processes including the packaging of genetic material in eukaryotes. For example, chromatin fibers are compacted in high ionic strength environments as are the minimal unit of packaging in chromatin, nucleosome core particles (NCPs). Furthermore, ionic strength is known to modulate several aspects of NCP dynamics including transient unwrapping of DNA from the histone protein core, nucleosome gaping, and intra- and internucleosomal interactions of the N-terminal histone tails. Changes in NCP structure may also impact interactions of transcriptional, repair, and other cellular machinery with nucleosomal DNA. One repair process, base excision repair (BER), is impacted by NCP structure and may be further influenced by changes in ionic strength. Here we examine the effects of ionic strength on the initiation of BER using biochemical assays. Using a population of NCPs containing uracil (U) at dozens of geometric locations, excision of U by single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is assessed at higher and lower ionic strengths. SMUG1 has increased excision activity in the lower ionic strength conditions. On duplex DNA, however, SMUG1 activity is largely unaffected by ionic strength except at short incubation times, suggesting that changes in SMUG1 activity are likely due to alterations in NCP structure and dynamics. These results allow us to further understand the cellular role of SMUG1 in a changing ionic environment and broadly contribute to the understanding of BER on chromatin and genomic stability.

DNA Base Excision Repair Intermediates Influence Duplex-Quadruplex Equilibrium.

Although genomic DNA is predominantly duplex under physiological conditions, particular sequence motifs can favor the formation of alternative secondary structures, including the G-quadruplex. These structures can exist within gene promoters, telomeric DNA, and regions of the genome frequently found altered in human cancers. DNA is also subject to hydrolytic and oxidative damage, and its local structure can influence the type of damage and its magnitude. Although the repair of endogenous DNA damage by the base excision repair (BER) pathway has been extensively studied in duplex DNA, substantially less is known about repair in non-duplex DNA structures. Therefore, we wanted to better understand the effect of DNA damage and repair on quadruplex structure. We first examined the effect of placing pyrimidine damage products uracil, 5-hydroxymethyluracil, the chemotherapy agent 5-fluorouracil, and an abasic site into the loop region of a 22-base telomeric repeat sequence known to form a G-quadruplex. Quadruplex formation was unaffected by these analogs. However, the activity of the BER enzymes were negatively impacted. Uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1) were inhibited, and apurinic/apyrimidinic endonuclease 1 (APE1) activity was completely blocked. Interestingly, when we performed studies placing DNA repair intermediates into the strand opposite the quadruplex, we found that they destabilized the duplex and promoted quadruplex formation. We propose that while duplex is the preferred configuration, there is kinetic conversion between duplex and quadruplex. This is supported by our studies using a quadruplex stabilizing molecule, pyridostatin, that is able to promote quadruplex formation starting from duplex DNA. Our results suggest how DNA damage and repair intermediates can alter duplex-quadruplex equilibrium.

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.

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.

Competition for DNA binding between the genome protector replication protein A and the genome modifying APOBEC3 single-stranded DNA deaminases.

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.

A novel Family V uracil DNA glycosylase from Sulfolobus islandicus REY15A.

Uracil DNA glycosylase (UDG) can excise uracil from DNA, thus playing an essential role in counteracting mutations. The genome of the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A encodes one putative Family V UDG (Sis-UDGV). Herein, we provide evidence that Sis-UDGV is a bi-functional glycosylase that can not only excise uracil from DNA, but cleave the generated apurinic/apyrimidinic (AP) site, which differs from other reported mono-functional Family V UDG homologs. Intriguingly, the enzyme can cleave DNA containing an AP site, thus suggesting that it might be involved in AP site repair. Biochemical data demonstrate that Sis-UDGV displays maximum activity for uracil removal at 45 °C ∼ 65 oC and at pH 8.0 ∼ 9.0. Furthermore, Sis-UDGV displays a substrate preference for uracil-containing ssDNA over uracil-containing dsDNA, but has no activity and weak activity for excising hypoxanthine from ssDNA and dsDNA, respectively. Importantly, we dissected the roles of seven conserved residues in Sis-UDGV by mutational analyses, demonstrating that residues D91, E117, E128, H167 and R192 are essential for catalysis. To our knowledge, it is the first report on the novel Family V UDG from Archaea with bi-functionality that harbors glycosylase/AP lyase activity.

SMUG1 regulates fat homeostasis leading to a fatty liver phenotype in mice.

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.

DNAzyme-powered cascade DNA walkers for sensitive detection of uracil DNA glycosylase activity.

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 × 10-5 U mL-1. The cascade walkers offer a new method for sensitively analyzing glycosylase.