DNA Adenine Methyltransferase Research Articles

N6-methyloxyadenine-mediated detoxification and ferroptosis confer a trade-off between multi-fungicide resistance and fitness.

Multi-fungicide resistance (MFR) is a serious environmental problem, which results in the excessive use of fungicides. Fitness penalty, as a common phenomenon in MFR, can partially counteract the issue of resistance due to the weakened vigor of MFR pathogens. Their underlying mechanism and relationship remain unexplained. By Oxford Nanopore Technologies sequencing and dot blot, we found that N6-methyloxyadenine (6mA) modification, the dominate epigenetic marker in Phytophthora capsici, was significantly altered after MFR emerged. Among the differently methylated genes, PcGSTZ1 could efficiently detoxify SYP-14288, a novel uncoupler, through complexing the fungicide with glutathione and induce MFR. Interestingly, PcGSTZ1 overexpression was induced by elevated 6mA levels and chromatin accessibility to its genomic loci. Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in P. capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. In conclusion, the findings provide new insights into the biological role of 6mA as well as the mechanisms underlying the trade-off between MFR and fitness. These could also benefit disease control through the blockade of the epigenetic axis to resensitize resistant isolates.IMPORTANCEN6-methyloxyadenine (6mA) modification on DNA is correlated with tolerance under different stress in prokaryotes. However, the role of 6mA in eukaryotes remains poorly understood. Our current study reveals that DNA adenine methyltransferase 1 (DAMT1)-mediated 6mA modification at the upstream region of GST zeta 1 (GSTZ1) is elevated in the resistant strain. This elevation promotes the detoxification uncoupler and induces multifungicide resistance (MFR). Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in Phytophthora capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici. Overall, our findings uncover an innovative mechanism underlying 6mA modification in regulating PcGSTZ1 transcription and the ferroptosis pathway in P. capsici.

A prophage encoded ribosomal RNA methyltransferase regulates the virulence of Shiga-toxin-producing Escherichia coli (STEC).

Shiga toxin (Stx) released by Shiga toxin producing Escherichia coli (STEC) causes life-threatening illness. Its production and release require induction of Stx-encoding prophage resident within the STEC genome. We identified two different STEC strains, PA2 and PA8, bearing Stx-encoding prophage whose sequences primarily differ by the position of an IS629 insertion element, yet differ in their abilities to kill eukaryotic cells and whose prophages differ in their spontaneous induction frequencies. The IS629 element in ϕPA2, disrupts an ORF predicted to encode a DNA adenine methyltransferase, whereas in ϕPA8, this element lies in an intergenic region. Introducing a plasmid expressing the methyltransferase gene product into ϕPA2 bearing-strains increases both the prophage spontaneous induction frequency and virulence to those exhibited by ϕPA8 bearing-strains. However, a plasmid bearing mutations predicted to disrupt the putative active site of the methyltransferase does not complement either of these defects. When complexed with a second protein, the methyltransferase holoenzyme preferentially uses 16S rRNA as a substrate. The second subunit is responsible for directing the preferential methylation of rRNA. Together these findings reveal a previously unrecognized role for rRNA methylation in regulating induction of Stx-encoding prophage.

Integrative DNA methylome and transcriptome analysis reveals DNA adenine methylation is involved in Salmonella enterica Typhimurium response to oxidative stress.

Salmonella could survive and replicate in macrophages, where it encounters multiple stresses. Deficiency of a DNA adenine methyltransferase impairs the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in hydrogen peroxide. To investigate whether DNA methylation is involved in the expression of oxidative stress-responsive genes, we combined RNA-seq and single-molecule real-time sequencing to integrate transcriptome and methylome analysis. Here, we show that (i) the entire amount of m6A GATC remains stable during oxidative stress; (ii) no significant association is observed between DNA methylation and transcription level in most genes; (iii) coincidence of level change between transcription and m6A GATC in the regulatory regions is identified in 49 genes under oxidative stress. Some of them are known to contribute to bacterial defenses against oxidative stress through reducing H2O2 levels, directing aberrant protein product degradation and inducing outer membrane protein expression. Specifically, the transcription level of ahpCF is negatively correlated to the m6A GATC level in its regulatory region. The transcription of smpB is elevated along with the decrease in m6A level at position 2,879,954 on the minus strand. In contrast, mRNA levels of STM14_2773 and ycfR are positively correlated with m6A GATC content in their regulatory regions. Highly stable DNA methylome and coupled change of m6A GATC with gene expression in specific positions suggest that DNA methylation homeostasis at genome-wide and plasticity in specific regions are crucial for bacterial response to oxidative stress. These findings provide new insights into the epigenetic regulatory mechanisms of gene expression in S. Typhimurium in the host microenvironment.IMPORTANCEThe intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) comes across a wide variety of stresses from entry to dissemination, such as reactive oxygen species. To adapt itself to oxidative stress, Salmonella must adopt various and complex strategies. In this study, we revealed that DNA adenine methyltransferase was essential for S. Typhimurium to survive in hydrogen peroxide. We then screened out oxidative stress-responsive genes that were potentially regulated by DNA methylation in S. Typhimurium. Our results show that the DNA methylome is highly stable throughout the genome, and the coupled change of m6A GATC with gene expression is identified in only a few positions, which suggests the complexity of the DNA methylation and gene expression regulation networks. The results may shed light on our understanding of m6A-mediated gene expression regulation in bacteria.

High fidelity DNA strand-separation is the major specificity determinant in DNA methyltransferase CcrM's catalytic mechanism.

Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure. The bacterial DNA adenine methyltransferase, CcrM, recognizes 5'GANTC'3 sequences through a DNA strand-separation mechanism with unusually high selectivity. To explore this novel recognition mechanism, we incorporated Pyrrolo-dC into cognate and noncognate DNA to monitor the kinetics of strand-separation and used tryptophan fluorescence to follow protein conformational changes. Both signals are biphasic and global fitting showed that the faster phase of DNA strand-separation was coincident with the protein conformational transition. Non-cognate sequences did not display strand-separation and methylation was reduced>300-fold, providing evidence that strand-separation is a major determinant of selectivity. Analysis of an R350A mutant showed that the enzyme conformational step can occur without strand-separation, so the two events are uncoupled. A stabilizing role for the methyl-donor (SAM) is proposed; the cofactor interacts with a critical loop which is inserted between the DNA strands, thereby stabilizing the strand-separated conformation. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural features implicated in strand-separation, which are found widely dispersed across many bacterial phyla, including human and animal pathogens, and some Eukaryotes.

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells.

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.

Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

Comparative Study of Adenosine Analogs as Inhibitors of Protein Arginine Methyltransferases and a Clostridioides difficile-Specific DNA Adenine Methyltransferase.

S-Adenosyl-l-methionine (SAM) analogs are adaptable tools for studying and therapeutically inhibiting SAM-dependent methyltransferases (MTases). Some MTases play significant roles in host-pathogen interactions, one of which is Clostridioides difficile-specific DNA adenine MTase (CamA). CamA is needed for efficient sporulation and alters persistence in the colon. To discover potent and selective CamA inhibitors, we explored modifications of the solvent-exposed edge of the SAM adenosine moiety. Starting from the two parental compounds (6e and 7), we designed an adenosine analog (11a) carrying a 3-phenylpropyl moiety at the adenine N6-amino group, and a 3-(cyclohexylmethyl guanidine)-ethyl moiety at the sulfur atom off the ribose ring. Compound 11a (IC50 = 0.15 μM) is 10× and 5× more potent against CamA than 6e and 7, respectively. The structure of the CamA-DNA-inhibitor complex revealed that 11a adopts a U-shaped conformation, with the two branches folded toward each other, and the aliphatic and aromatic rings at the two ends interacting with one another. 11a occupies the entire hydrophobic surface (apparently unique to CamA) next to the adenosine binding site. Our work presents a hybrid knowledge-based and fragment-based approach to generating CamA inhibitors that would be chemical agents to examine the mechanism(s) of action and therapeutic potentials of CamA in C. difficile infection.

DamID-seq: A Genome-WideDNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fusedto a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-targetgene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Herewe provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.

Tetrahedral DNA nanostructure-corbelled click chemistry-based large-scale assembly of nanozymes for ratiometric fluorescence assay of DNA methyltransferase activity.

Ligation efficiency in a surface-based DNA click chemistry (CuAAC) reaction is extremely restricted by the orientation and density of probes arranged on a heterogeneous surface. Herein, we engineer DNA tetrahedral nanostructure (DTN)-corbelled click chemistry to trigger a hybridization chain reaction (HCR) assembling a large-scale of nanozymes for ratiometric fluorescence detection of DNA adenine methyltransferase (Dam). In this study, a DNA tetrahedron structure with an alkynyl modifying pendant DNA probe (Alk-DTN) is designed and assembled on a magnetic bead (MB) as a scaffold for click chemistry. When a CuO NP-encoded magnetic nanoparticle (CuO-MNP) substrate was methylated by Dam, CuO NPs were released and turned into a mass of Cu+. The Cu+ droves azido modifying lDNA (azide-lDNA) to connect with the Alk-DTN probe on the MB through the click reaction, forming an intact primer to initiate the HCR. The HCR product, a rigid structure double-stranded DNA, periodically assembles glucose oxidase mimicking gold nanoparticles (GNPs) into a large-scale of nanozymes for catalyzing the oxidation of glucose to H2O2. NH2-MIL-101 MOFs, a fluorescent indicator and a biomimetic catalyst, activated the product H2O2 to oxidize o-phenylenediamine (oPD) into visually detectable 2,3-diaminophenazine (DAP). The change of the signal ratio between DAP and NH2-MIL-101 is proportional to the methylation event corresponding to the MTase activity. In this study, the DTN enhances the efficiency of the surface-based DNA click reaction and maintains the catalytic activities of gold nanoparticle nanozymes due to the intrinsic nature of mechanical rigidity and well-controlled orientation and well-adjusted size. Large-scale assembly of nanozymes circumvents the loss of natural enzyme activity caused by chemical modification and greatly improves the amplification efficiency. The proposed biosensor displayed a low detection limit of 0.001 U mL-1 for Dam MTase due to multiple amplification and was effective in real samples and methylation inhibitor screening, providing a promising modular platform for bioanalysis.

Antimicrobial resistance in Klebsiella pneumoniae: identification of bacterial DNA adenine methyltransferase as a novel drug target from hypothetical proteins using subtractive genomics.

Klebsiella pneumoniae is a gram-negative bacterium that is known for causing infection innosocomial settings. As reported by the World Health Organization, carbapenem-resistantEnterobacteriaceae, a category that includes K. pneumoniae, are classified as an urgentthreat, and the greatest concern is that these bacterial pathogens may acquire genetictraits that make them resistant towards antibiotics. The last class of antibiotics, carbapenems, are not able to combat these bacterial pathogens, allowing them to clonally expandantibiotic-resistant strains. Most antibiotics target essential pathways of bacterial cells;however, these targets are no longer susceptible to antibiotics. Hence, in our study, we focused on a hypothetical protein in K. pneumoniae that contains a DNA methylation proteindomain, suggesting a new potential site as a drug target. DNA methylation regulates theattenuation of bacterial virulence. We integrated computational-aided drug design by using a bioinformatics approach to perform subtractive genomics, virtual screening, and fingerprint similarity search. We identified a new potential drug, koenimbine, which could bea novel antibiotic.

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 μM and KD ∼ 0.2 μM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection

DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3’ end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10−4 U/mL and a broad detection range from 2 × 10−4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.

Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA

In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen. A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studies revealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possible interaction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with subsequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addition, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interaction partners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in the milligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gel electrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. These analyses did not provide evidence of interaction between AamA and purified proteins. Solution structures of RibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA. While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA to NrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transient interaction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferase activity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts to identify the interaction network of AamA.

A Hidden Markov Model for predicting the methylome of the DNA adenine methyltransferase in bacterial genome

Epigenetic mechanisms such as DNA adenine methyltransferase (DAM) in the bacterial genome play important roles in the cell systems to ensure the control and regulation of cellular functioning. The localization of the DAM sites in bacterial genome is still a challenge due to the complexity of experimental tests. In this study, we propose a new computational model to predict DAM sites in the bacterial genome using the Hidden Markov Model (HMM) and emission process, henceforth labeled as DAM-HMM. The proposed DAM-HMM model is generated by simulating the Single-Molecule Real-Time Sequencing (SMRT) data. The model is validated using artificial methylation sequences and then compared with the methylation results of the E. coli full genome sequenced by SMRT method. The validation results of the DAM-HMM model show a high sensitivity of 90% and specificity of 86% to the DAM methylome which is consistent with the previous experimental studies. In addition, the DAM-HMM model simulation of the whole bacterial genome methylation patterns as applied to some Desulvibrio members reveals the presence of regions with high rate of methylation.

Multiomic chromatin and transcription profiling with EpiDamID

DamID maps protein-genome interactions using DNA adenine methyltransferase tethered to individual chromatin proteins. In a recent issue of Molecluar Cell, Rang et al. introduce EpiDamID, a powerful extension of DamID suitable for mapping histone marks while simultaneously measuring mRNA levels in single cells.

Genome-wide lone strand adenine methylation in Deinococcus radiodurans R1: Regulation of gene expression through DR0643-dependent adenine methylation.

DNA methylation is a covalent modification of adenine or cytosine in the genome of an organism and is found in diverse microbes including the radiation resistant bacterium Deinococcus radiodurans R1. Although earlier findings have confirmed repression or de-repression of certain genes in adenine methyltransferase (DR_0643/Dam1DR) deficient D. radiodurans mutant however, the overall regulatory aspects of Dam1DR-mediated adenine methylation remain mostly unexplored. In the present study, we compared the genome-wide methylome and the corresponding transcriptome of D. radiodurans WT and Δdam1 mutant to explore the correlation between methylation and gene expression. In D. radiodurans, deletion of DR_0643 ORF (Δdam1) led to hypomethylation of 512 genes resulting in differential expression of 168 genes (99 genes are upregulated and 69 genes are downregulated). The modification patterns deduced for Dam1DR (DR_0643) and Dam2DR (DR_2267) were non-palindromic and atypical. Moreover, we observed methylation at opportunistic sites that show adenine methylation only in D. radiodurans Δdam1 and not in D. radiodurans WT. Correlation between the methylome and transcriptome suggests that hypomethylation at Dam1DR specific sites had both negative as well as a positive effects on gene expression. Pathways such as amino acid metabolism, transport, oxidative phosphorylation, quorum sensing, signal transduction, two-component system, glycolysis/gluconeogenesis, TCA cycle, glyoxylate and dicarboxylate metabolism were modulated by Dam1DR-mediated adenine methylation in D. radiodurans. Processes such as DNA repair, recombination, ATPase and transmembrane transporter activity were enriched when Dam1DR mutant was subjected to radiation stress. We further evaluated the molecular interactions and mode of binding between Dam1DR protein and S-adenosyl methionine using molecular docking followed by MD simulation. To get a better insight into the methylation mechanism, the Dam1DR-SAM complex was also docked with a DNA molecule to elucidate DNA-Dam1DR structural interaction during methyl-group transfer reaction. In summary, our work presents comprehensive and integrative approaches to investigate both functional and structural aspects of DNA adenine methyltransferase (Dam1DR) in D. radiodurans biology.

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor.

DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

Effects of lamina associated domain on the three dimensional shapes of chromosome territories and nucleoplasmic cavities in cell nucleus

Chromatin can interact with lamin of the cellular nuclear envelope and plays important roles in 3D genome organization. Lamina Associated Domains (LAD) are regions on the chromatin where interaction with proteins in the lamin occur. Recent studies of DNA adenine methyltransferase identification (DamID) assay have mapped out LAD locations genomewide. Here we study the effects of LAD and lamin interactions on chromatin territories, chromatin shapes, and nucleopasmic cavities.

Repurposing epigenetic inhibitors to target the Clostridioides difficile-specific DNA adenine methyltransferase and sporulation regulator CamA

ABSTRACT Epigenetically targeted therapeutic development, particularly for SAM-dependent methylations of DNA, mRNA and histones has been proceeding rapidly for cancer treatments over the past few years. However, this approach has barely begun to be exploited for developing new antibiotics, despite an overwhelming global need to counter antimicrobial resistance. Here, we explore whether SAM analogues, some of which are in (pre)clinical studies as inhibitors of human epigenetic enzymes, can also inhibit Clostridioides difficile-specific DNA adenine methyltransferase (CamA), a sporulation regulator present in all C. difficile genomes sequenced to date, but found in almost no other bacteria. We found that SGC0946 (an inhibitor of DOT1L), JNJ-64619178 (an inhibitor of PRMT5) and SGC8158 (an inhibitor of PRMT7) inhibit CamA enzymatic activity in vitro at low micromolar concentrations. Structural investigation of the ternary complexes of CamA-DNA in the presence of SGC0946 or SGC8158 revealed conformational rearrangements of the N-terminal arm, with no apparent disturbance of the active site. This N-terminal arm and its modulation of exchanges between SAM (the methyl donor) and SAH (the reaction product) during catalysis of methyl transfer are, to date, unique to CamA. Our work presents a substantial first step in generating potent and selective inhibitors of CamA that would serve in the near term as chemical probes to investigate the cellular mechanism(s) of CamA in controlling spore formation and colonization, and eventually as therapeutic antivirulence agents useful in treating C. difficile infection.

Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix

Clostridioides difficile infections are an urgent medical problem. The newly discovered C.difficileadenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-l-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.