Programmable DNA pyrimidine base editing via engineered uracil-DNA glycosylase.

DNA base editing technologies predominantly utilize engineered deaminases, limiting their ability to edit thymine and guanine directly. In this study, we successfully achieve base editing of both cytidine and thymine by leveraging the translesion DNA synthesis pathway through the engineering of uracil-DNA glycosylase (UNG). Employing structure-based rational design, exploration of homologous proteins, and mutation screening, we identify a Deinococcus radiodurans UNG mutant capable of effectively editing thymine. When fused with the nickase Cas9, the engineered DrUNG protein facilitates efficient thymine base editing at endogenous sites, achieving editing efficiencies up to 55% without enrichment and exhibiting minimal cellular toxicity. This thymine base editor (TBE) exhibits high editing specificity and significantly restores IDUA enzyme activity in cells derived from patients with Hurler syndrome. TBEs represent efficient, specific, and low-toxicity approaches to base editing with potential applications in treating relevant diseases.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SIGNIFICANCE: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

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.

Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides.

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase ß. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

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.

Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

BACKGROUND: Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. OBJECTIVE: This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. METHODS: The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. RESULTS: This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. CONCLUSION: We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

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.

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.

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.

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.

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.

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.

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.

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.

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.