Статті в журналах: "DNA MTase"

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Zhu, Chen, Shuting Zhang, Chengzhe Zhou, Lan Chen, Haifeng Fu, Xiaozhen Li, Yuling Lin, Zhongxiong Lai, and Yuqiong Guo. "Genome-wide investigation and transcriptional analysis of cytosine-5 DNA methyltransferase and DNA demethylase gene families in tea plant (Camellia sinensis) under abiotic stress and withering processing." PeerJ 8 (January 14, 2020): e8432. http://dx.doi.org/10.7717/peerj.8432.

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DNA methylation is a highly conserved epigenetic modification involved in many biological processes, including growth and development, stress response, and secondary metabolism. In the plant kingdom, cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes have been identified in some plant species. However, to the best of our knowledge, no investigator has focused on the identification and analysis of C5-MTase and dMTase genes in tea plants (Camellia sinensis) based on genome-wide levels. In this study, eight CsC5-MTases and four dMTases were identified in tea plants. These CsC5-MTase genes were divided into four subfamilies, including CsMET, CsCMT, CsDRM and CsDNMT2. The CsdMTase genes can be classified into CsROS, CsDME and CsDML. Based on conserved domain analysis of these genes, the gene loss and duplication events occurred during the evolution of CsC5-MTase and CsdMTase. Furthermore, multiple cis-acting elements were observed in the CsC5-MTase and CsdMTase, including light responsiveness, phytohormone responsiveness, stress responsiveness, and plant growth and development-related elements. Then, we investigated the transcript abundance of CsC5-MTase and CsdMTase under abiotic stress (cold and drought) and withering processing (white tea and oolong tea). Notably, most CsC5-MTases, except for CsCMT1 and CsCMT2, were significantly downregulated under abiotic stress, while the transcript abundance of all four CsdMTase genes was significantly induced. Similarly, the same transcript abundance of CsC5-MTase and CsdMTase was found during withering processing of white tea and oolong tea, respectively. In total, our findings will provide a basis for the roles of CsC5-MTase and CsdMTase in response to abiotic stress and the potential functions of these two gene families in affecting tea flavor during tea withering processing.

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2

Li, Jiang, Caili Li, and Shanfa Lu. "Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza." PeerJ 6 (March 5, 2018): e4461. http://dx.doi.org/10.7717/peerj.4461.

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Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants.

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Ginibre, Nadège, Ludovic Legrand, Victoria Bientz, Jean-Claude Ogier, Anne Lanois, Sylvie Pages, and Julien Brillard. "Diverse Roles for a Conserved DNA-Methyltransferase in the Entomopathogenic Bacterium Xenorhabdus." International Journal of Molecular Sciences 23, no. 19 (October 9, 2022): 11981. http://dx.doi.org/10.3390/ijms231911981.

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In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria.

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4

Wang, Yuehua, Yingli Han, Fangyu Zhou, Tingting Fan, and Feng Liu. "Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe." Biosensors 12, no. 8 (July 26, 2022): 569. http://dx.doi.org/10.3390/bios12080569.

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DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBRTM Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5–110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.

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5

Shi, Lisha, Huimin Shen, Jiawei Liu, Hongmin Hu, Hongyan Tan, Xiulian Yang, Lianggui Wang, and Yuanzheng Yue. "Exploration of the Potential Transcriptional Regulatory Mechanisms of DNA Methyltransferases and MBD Genes in Petunia Anther Development and Multi-Stress Responses." Genes 13, no. 2 (February 8, 2022): 314. http://dx.doi.org/10.3390/genes13020314.

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Cytosine-5 DNA methyltransferases (C5-MTases) and methyl-CpG-binding-domain (MBD) genes can be co-expressed. They directly control target gene expression by enhancing their DNA methylation levels in humans; however, the presence of this kind of cooperative relationship in plants has not been determined. A popular garden plant worldwide, petunia (Petunia hybrida) is also a model plant in molecular biology. In this study, 9 PhC5-MTase and 11 PhMBD proteins were identified in petunia, and they were categorized into four and six subgroups, respectively, on the basis of phylogenetic analyses. An expression correlation analysis was performed to explore the co-expression relationships between PhC5-MTases and PhMBDs using RNA-seq data, and 11 PhC5-MTase/PhMBD pairs preferentially expressed in anthers were identified as having the most significant correlations (Pearson’s correlation coefficients > 0.9). Remarkably, the stability levels of the PhC5-MTase and PhMBD pairs significantly decreased in different tissues and organs compared with that in anthers, and most of the selected PhC5-MTases and PhMBDs responded to the abiotic and hormonal stresses. However, highly correlated expression relationships between most pairs were not observed under different stress conditions, indicating that anther developmental processes are preferentially influenced by the co-expression of PhC5-MTases and PhMBDs. Interestingly, the nuclear localization genes PhDRM2 and PhMBD2 still had higher correlations under GA treatment conditions, implying that they play important roles in the GA-mediated development of petunia. Collectively, our study suggests a regulatory role for DNA methylation by C5-MTase and MBD genes in petunia anther maturation processes and multi-stress responses, and it provides a framework for the functional characterization of C5-MTases and MBDs in the future.

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6

Bheemanaik, Shivakumara, Yeturu V. R. Reddy, and Desirazu N. Rao. "Structure, function and mechanism of exocyclic DNA methyltransferases." Biochemical Journal 399, no. 2 (September 27, 2006): 177–90. http://dx.doi.org/10.1042/bj20060854.

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DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an ‘AdoMet-dependent MTase fold’. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein–DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.

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Hiraoka, Satoshi, Tomomi Sumida, Miho Hirai, Atsushi Toyoda, Shinsuke Kawagucci, Taichi Yokokawa, and Takuro Nunoura. "Diverse DNA modification in marine prokaryotic and viral communities." Nucleic Acids Research 50, no. 3 (January 21, 2022): 1531–50. http://dx.doi.org/10.1093/nar/gkab1292.

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Abstract DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent ‘metaepigenomic’ analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.

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Zhang, Yufeng, Chunxiao Liu, Xiaoyang Xu, Jialiang Kan, Hui Li, Jing Lin, Zongming Cheng, and Youhong Chang. "Comprehensive Analysis of the DNA Methyltransferase Genes and Their Association with Salt Response in Pyrus betulaefolia." Forests 14, no. 9 (August 30, 2023): 1751. http://dx.doi.org/10.3390/f14091751.

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DNA methylation plays an indispensable role in genome stability, regulation of gene expression and plant stress response. It is mediated by DNA methyltransferases (MTases). Twelve putative MTases of P. betulaefolia were identified and were classified into MET1, CMT, DRM2 and Dnmt2 groups based on the organization of various characteristic domains. Three pairs of paralogous genes were identified with the Ka/Ks ratio varied from 0.232 for PbeMET1a and PbeMET1b to 0.251 for PbeCMT2 and PbeCMT3, respectively. In addition, the Ka/Ks ratio for nine pairs of orthologous gene pairs between P. betulaefolia and apple were varied from 0.053 for PbeDRM3 and MD17G1031900 to 0.278 for PbeDnmt2b and MD15G1120500, respectively. All the 12 members of MTase genes were located on nine chromosomes out of 17 P. betulaefolia chromosomes with highly conserved protein sequence structures. The isoelectric points (pI) of MTases ranged from 4.74 to 7.24, while molecular weight varied from 35.99 to 174.32. The expression profiles of MTase and other salt-responsive genes under salt stress treatment revealed their important roles involved in salt response in P. betulaefolia. Furthermore, three selected salt-responsive genes (PbeNHX2.1, PbeCBL2 and PbeAKT2) were found altered in methylation level of promoters (which contain CpG islands) under salt stress. Especially, the PbeAKT2 promoter regions showed high CHG and CHH methylation types. Our study provided a genome-wide survey of the MTase gene family and highlighted their roles in salt response. These results also provided an effective way for the breeding and improvement of salt-tolerant pear varieties.

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9

Oerum, Stephanie, Vincent Meynier, Marjorie Catala, and Carine Tisné. "A comprehensive review of m6A/m6Am RNA methyltransferase structures." Nucleic Acids Research 49, no. 13 (May 22, 2021): 7239–55. http://dx.doi.org/10.1093/nar/gkab378.

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Abstract Gene expression is regulated at many levels including co- or post-transcriptionally, where chemical modifications are added to RNA on riboses and bases. Expression control via RNA modifications has been termed ‘epitranscriptomics’ to keep with the related ‘epigenomics’ for DNA modification. One such RNA modification is the N6-methylation found on adenosine (m6A) and 2′-O-methyladenosine (m6Am) in most types of RNA. The N6-methylation can affect the fold, stability, degradation and cellular interaction(s) of the modified RNA, implicating it in processes such as splicing, translation, export and decay. The multiple roles played by this modification explains why m6A misregulation is connected to multiple human cancers. The m6A/m6Am writer enzymes are RNA methyltransferases (MTases). Structures are available for functionally characterized m6A RNA MTases from human (m6A mRNA, m6A snRNA, m6A rRNA and m6Am mRNA MTases), zebrafish (m6Am mRNA MTase) and bacteria (m6A rRNA MTase). For each of these MTases, we describe their overall domain organization, the active site architecture and the substrate binding. We identify areas that remain to be investigated, propose yet unexplored routes for structural characterization of MTase:substrate complexes, and highlight common structural elements that should be described for future m6A/m6Am RNA MTase structures.

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10

Zhang, Weiting, Xiaolong Zu, Yanling Song, Zhi Zhu, and Chaoyong James Yang. "Detection of DNA methyltransferase activity using allosteric molecular beacons." Analyst 141, no. 2 (2016): 579–84. http://dx.doi.org/10.1039/c5an01763g.

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Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity.

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Vertino, P. M., R. W. Yen, J. Gao, and S. B. Baylin. "De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase." Molecular and Cellular Biology 16, no. 8 (August 1996): 4555–65. http://dx.doi.org/10.1128/mcb.16.8.4555.

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Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

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Bheemanaik, Shivakumara, Janusz M. Bujnicki, Valakunja Nagaraja, and Desirazu N. Rao. "Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase." Biological Chemistry 387, no. 5 (May 1, 2006): 515–23. http://dx.doi.org/10.1515/bc.2006.067.

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AbstractKpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5′-GGTACC-3′ and transfers the methyl group fromS-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation andin vitrorestriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

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Tsukamoto, Yumiko, Toshiki Tamura, Yumi Maeda, Kensuke Miyake, and Manabu Ato. "6-Methyladenine may enhance immunostimulatory activity of bacterial genomic DNA." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 226.17. http://dx.doi.org/10.4049/jimmunol.204.supp.226.17.

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Abstract Background and Aim CpG motifs in DNA sequences are recognized by TLR9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine (5mC)) and modified adenine bases (6-methyladenine (6mA)). It is well-known that 5mC inhibits immune activation by CpG DNA; however, it is still unclear whether 6mA inhibits immune activation by CpG DNA. In this study, we tried to examine whether the existence of 6mA affected the immunostimulatory activity of gDNA. Materials and Methods We focused on adenine methyltransferases (MTases) expressed by Mycobacterium bovis BCG (BCG). Each adenine MTases has specific target sites. Therefore, we used oligodeoxynucleotides (ODNs) with 6mA on the target sequences of MTases from BCG. We stimulated murine bone-marrow derived macrophages (BMDMs) with ODNs harboring 6mA or ODNs without 6mA. We then compared the immunostimulatory activity of ODNs with 6mA to ODNs without 6mA using. We also developed adenine MTase-deficient BCG. We analyzed whether 6mA on gDNA from BCG is involved in the immunostimulatory activity of BCG gDNA. Results Our results showed that 6mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced IL-12p40 production from BMDMs stimulated with CpG DNA. We also observed that gDNA from mamA-deficient BCG induced less IL-12p40 from BMDMs than gDNA from mamA-intact BCG. Conclusion We concluded from these results that adenine methylation on ODNs may enhance immune activity induced by bacterial gDNA.

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14

Numata, M., T. Ono, and S. Iseki. "Expression and localization of the mRNA for DNA (cytosine-5)- methyltransferase in mouse seminiferous tubules." Journal of Histochemistry & Cytochemistry 42, no. 9 (September 1994): 1271–76. http://dx.doi.org/10.1177/42.9.8064134.

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DNA (cytosine-5)-methyltransferase (DNA MTase) is the only enzyme known to be involved in the methylation of mammalian DNA. Although the expression of DNA MTase gene is abundant in the testis, little is known about the role of this enzyme during spermatogenesis. We examined the distribution of DNA MTase mRNA in mouse testis by in situ hybridization histochemistry with an oligonucleotide probe. The mRNA signal was observed in the seminiferous tubules and was localized predominantly in spermatogonia and spermatocytes, particularly during the earlier steps of meiotic prophase I, with maximal intensity in the early pachytene cells. These results suggest some significant role for DNA MTase in spermatogenesis.

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Vitkute, Jolanta, Kornelijus Stankevicius, Giedre Tamulaitiene, Zita Maneliene, Albertas Timinskas, Douglas E. Berg, and Arvydas Janulaitis. "Specificities of Eleven Different DNA Methyltransferases of Helicobacter pylori Strain 26695." Journal of Bacteriology 183, no. 2 (January 15, 2001): 443–50. http://dx.doi.org/10.1128/jb.183.2.443-450.2001.

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ABSTRACT Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome ofHelicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m4C, m5C, and m6A.

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Banerjee, Sanjib, and Rukhsana Chowdhury. "An orphan DNA (cytosine-5-)-methyltransferase in Vibrio cholerae." Microbiology 152, no. 4 (April 1, 2006): 1055–62. http://dx.doi.org/10.1099/mic.0.28624-0.

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5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an ‘orphan’ or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 5′ C in the degenerate sequence 5′-RCCGGY-3′. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

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Bheemanaik, Shivakumara, Srivani Sistla, Vinita Krishnamurthy, Sampath Arathi, and Narasimha Rao Desirazu. "Kinetics of Methylation by EcoP1I DNA Methyltransferase." Enzyme Research 2010 (July 15, 2010): 1–14. http://dx.doi.org/10.4061/2010/302731.

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EcoP1I DNA MTase (M.EcoP1I), an N6-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg2+ for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn2+ and Ca2+ other than Mg2+ with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [I358D(x)n…ExK401 and D600xDxD604 motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

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Hu, Tingting, Changbei Ma, Ying Yan, and Junxiang Chen. "Detection of DNA Methyltransferase Activity via Fluorescence Resonance Energy Transfer and Exonuclease-Mediated Target Recycling." Biosensors 12, no. 6 (June 8, 2022): 395. http://dx.doi.org/10.3390/bios12060395.

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In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-methyladenine. In the method developed in this study, the methylated substrate is cleaved using Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is hybridized to the 3ʹ protruding end of the F-DNA probe to form a double-stranded DNA, which is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection, with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening, whereby such inhibitors can be identified for disease treatment.

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CIOFFI, Anna Valentina, Diana FERRARA, Maria Vittoria CUBELLIS, Francesco ANIELLO, Marcella CORRADO, Francesca LIGUORI, Alessandro AMOROSO, Laura FUCCI, and Margherita BRANNO. "An open reading frame in intron seven of the sea urchin DNA-methyltransferase gene codes for a functional AP1 endonuclease." Biochemical Journal 365, no. 3 (August 1, 2002): 833–40. http://dx.doi.org/10.1042/bj20011857.

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Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene. METEX expression is developmentally regulated, showing no correlation with DNA MTase expression. In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development. Two METEX cDNA clones (Met1 and Met2) that are different in the 3′ end have been isolated in a cDNA library screening. The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure. The protein, overexpressed in E. coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity. Therefore the open reading frame, present in intron 7 of the P. lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

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Bhattacharya, Tamanash, Danny W. Rice, John M. Crawford, Richard W. Hardy, and Irene L. G. Newton. "Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking." Viruses 13, no. 8 (July 27, 2021): 1464. http://dx.doi.org/10.3390/v13081464.

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Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. We, therefore, sought to understand the divergent regulation, function, and evolution of these orthologs. We describe the role of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2′s role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, we identify and describe potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. We identify specific codons within each ortholog that are under positive selection and find that they are restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, our findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs.

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Fomenkov, Alexey, Zhiyi Sun, Iain A. Murray, Cristian Ruse, Colleen McClung, Yoshiharu Yamaichi, Elisabeth A. Raleigh, and Richard J. Roberts. "Plasmid replication-associated single-strand-specific methyltransferases." Nucleic Acids Research 48, no. 22 (December 3, 2020): 12858–73. http://dx.doi.org/10.1093/nar/gkaa1163.

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Abstract Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.

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Kimura, Hiromichi, Toyokazu Takeda, Satoshi Tanaka, Tomoya Ogawa, and Kunio Shiota. "Expression of Rat DNA (cytosine-5) Methyltransferase (DNA MTase) in Rodent Trophoblast Giant Cells: Molecular Cloning and Characterization of Rat DNA MTase." Biochemical and Biophysical Research Communications 253, no. 2 (December 1998): 495–501. http://dx.doi.org/10.1006/bbrc.1998.9802.

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Hong, Lu, Jing Wan, Xiaojun Zhang, and Guangfeng Wang. "DNA–gold nanoparticles network based electrochemical biosensors for DNA MTase activity." Talanta 152 (May 2016): 228–35. http://dx.doi.org/10.1016/j.talanta.2016.01.026.

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Liu, Zhuoliang, Chunyang Lei, Honghua Deng, Guoyan Lu, Yan Huang, and Shouzhuo Yao. "Sensitive and versatile fluorescent enzymatic assay of nucleases and DNA methyltransferase based on a supercharged fluorescent protein." RSC Advances 6, no. 40 (2016): 34074–80. http://dx.doi.org/10.1039/c6ra02711c.

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Singh, I., Christine Beuck, Anupam Bhattacharya, Walburga Hecker, V. S. Parmar, E. Weinhold, and O. Seitz. "Abasic site stabilization by aromatic DNA base surrogates: High-affinity binding to a base-flipping DNA-methyltransferase." Pure and Applied Chemistry 76, no. 7-8 (January 1, 2004): 1563–70. http://dx.doi.org/10.1351/pac200476071563.

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DNA-methyltransferases catalyze the sequence-specific transfer of the methyl group of S-adenosylmethionine to target bases in genomic DNA. For gaining access to their target embedded within a double-helical structure, DNA-methyltransferases (DNA-MTases) rotate the target base out of the DNA helix. This base-flipping leads to the formation of an apparent abasic site. MTases such as cytosine-specific MHhaI and MHaeIII and also the repair enzyme uracil DNA glycosylase (UDG) insert amino acid side chains into the opened space and/or rearrange base-pairing. The adenine-specific DNA MTase MTaqI binds without amino acid insertion. This binding mode allows for a substitution of the orphaned thymine with larger DNA base surrogates without steric interference by inserted amino acid side chains. DNA containing pyrenyl, naphthyl, acenaphthyl, and biphenyl residues was tested in MTaqI binding studies. The synthesis of DNA building blocks required the formation of a C-glycosidic bond, which was established by using protected 1-chloro-2-deoxy- ribose as glycosyl donor and organocuprates as glycosyl acceptors. It is shown that all of the base surrogates enhanced the binding affinity to MTaqI. Incorporation of pyrene increased the binding affinity by a factor of 400. Interestingly, there is a correlation between the observed order of dissociation constants and the ability of a base surrogate to stabilize abasic sites in model duplexes.

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Liu, Yuanjian, Min Wei, Linqun Zhang, Wei Wei, Yuanjian Zhang, and Songqin Liu. "Evaluation of DNA methyltransferase activity and inhibition via chiroplasmonic assemblies of gold nanoparticles." Chemical Communications 51, no. 76 (2015): 14350–53. http://dx.doi.org/10.1039/c5cc05375g.

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Kumar, Ritesh, and Desirazu N. Rao. "A nucleotide insertion between two adjacent methyltransferases in Helicobacter pylori results in a bifunctional DNA methyltransferase." Biochemical Journal 433, no. 3 (January 14, 2011): 487–95. http://dx.doi.org/10.1042/bj20101668.

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Helicobacter pylori has a dynamic R-M (restriction–modification) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other H. pylori strains. Random mutations in R-M genes can result in non-functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA MTase (methyltransferase) genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM–hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and, when a similar mutation was made in hpyAVIAM–hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5-cytosine MTase domain and a C-terminal N6-adenine MTase domain was constructed. Mutations in the AdoMet (S-adenosylmethionine)-binding motif or in the catalytic motif of M.HpyAVIA or M.HpyAVIB selectively abrogated the C5-cytosine or N6-adenine methylation activity of M.HpyAVIA–M.HpyAVIB fusion protein. The present study highlights the ability of H. pylori to evolve genes with unique functions and thus generate variability. For organisms such as H. pylori, which have a small genome, these adaptations could be important for their survival in the hostile host environment.

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Wang, Hui-zhen, Minnie M. L. Wong, Desmond O'Toole, Mandy M. H. Mak, Rudolf S. S. Wu, and Richard Y. C. Kong. "Identification of a DNA Methyltransferase Gene Carried on a Pathogenicity Island-Like Element (VPAI) in Vibrio parahaemolyticus and Its Prevalence among Clinical and Environmental Isolates." Applied and Environmental Microbiology 72, no. 6 (June 2006): 4455–60. http://dx.doi.org/10.1128/aem.02095-05.

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ABSTRACT In this study we identified a putative virulence-associated DNA methyltransferase (MTase) gene carried on a novel 22.79-kb pathogenicity island-like element (VPAI) in V. parahaemolyticus. The V. parahaemolyticus MTase gene was shown by PCR to be prevalent (>98%) in pandemic thermostable direct hemolysin gene-positive isolates, which suggests that VPAI may confer unique virulence traits to pandemic strains of V. parahaemolyticus.

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Sedlackova, Eliska, Zuzana Bytesnikova, Eliska Birgusova, Pavel Svec, Amir M. Ashrafi, Pedro Estrela, and Lukas Richtera. "Label-Free DNA Biosensor Using Modified Reduced Graphene Oxide Platform as a DNA Methylation Assay." Materials 13, no. 21 (November 3, 2020): 4936. http://dx.doi.org/10.3390/ma13214936.

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This work reports the use of modified reduced graphene oxide (rGO) as a platform for a label-free DNA-based electrochemical biosensor as a possible diagnostic tool for a DNA methylation assay. The biosensor sensitivity was enhanced by variously modified rGO. The rGO decorated with three nanoparticles (NPs)—gold (AuNPs), silver (AgNPs), and copper (CuNPs)—was implemented to increase the electrode surface area. Subsequently, the thiolated DNA probe (single-stranded DNA, ssDNA−1) was hybridized with the target DNA sequence (ssDNA-2). After the hybridization, the double-stranded DNA (dsDNA) was methylated by M.SssI methyltransferase (MTase) and then digested via a HpaII endonuclease specific site sequence of CpG (5′-CCGG-3′) islands. For monitoring the MTase activity, differential pulse voltammetry (DPV) was used, whereas the best results were obtained by rGO-AuNPs. This assay is rapid, cost-effective, sensitive, selective, highly specific, and displays a low limit of detection (LOD) of 0.06 U·mL−1. Lastly, this study was enriched with the real serum sample, where a 0.19 U·mL−1 LOD was achieved. Moreover, the developed biosensor offers excellent potential in future applications in clinical diagnostics, as this approach can be used in the design of other biosensors.

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Liu, Yan-Ping, Qun Tang, Jie-Zhong Zhang, Li-Fei Tian, Pu Gao, and Xiao-Xue Yan. "Structural basis underlying complex assembly and conformational transition of the type I R-M system." Proceedings of the National Academy of Sciences 114, no. 42 (October 2, 2017): 11151–56. http://dx.doi.org/10.1073/pnas.1711754114.

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Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the “open” form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution “closed” structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

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Ji, Lijuan, Zhewei Cai, Yingdan Qian, Ping Wu, Hui Zhang, and Chenxin Cai. "Highly sensitive methyltransferase activity assay and inhibitor screening based on fluorescence quenching of graphene oxide integrated with the site-specific cleavage of restriction endonuclease." Chem. Commun. 50, no. 73 (2014): 10691–94. http://dx.doi.org/10.1039/c4cc04428b.

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Li, Xuemei, Ting Song, and Xilin Guo. "DNA methylation detection with end-to-end nanorod assembly-enhanced surface plasmon resonance." Analyst 140, no. 18 (2015): 6230–33. http://dx.doi.org/10.1039/c5an01015b.

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Wang, Li-juan, Xiao Han, Chen-chen Li, and Chun-yang Zhang. "Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase." Chemical Science 9, no. 28 (2018): 6053–61. http://dx.doi.org/10.1039/c8sc02215a.

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Lorincz, Matthew C., Dirk Schübeler, Shauna R. Hutchinson, David R. Dickerson, and Mark Groudine. "DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation." Molecular and Cellular Biology 22, no. 21 (November 1, 2002): 7572–80. http://dx.doi.org/10.1128/mcb.22.21.7572-7580.2002.

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ABSTRACT DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

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Li, Fang, Xiuming Wu, Mengmeng Gu, and Guang-Li Wang. "Label-free and highly sensitive detection of DNA adenine methylation methyltransferase through cathodic photoelectrochemistry." Analyst 146, no. 8 (2021): 2646–52. http://dx.doi.org/10.1039/d0an02438d.

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Negri, Alessandro, Olesia Werbowy, Ewa Wons, Simon Dersch, Rebecca Hinrichs, Peter L. Graumann, and Iwona Mruk. "Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies." Nucleic Acids Research 49, no. 7 (March 21, 2021): 3826–40. http://dx.doi.org/10.1093/nar/gkab183.

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Abstract Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

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Chen, Ying, and Hongchao Yi. "A glucometer-based strategy for sensitive DNA methyltransferase activity detection via a polymerization nicking reaction and enzyme amplification." Analytical Methods 9, no. 20 (2017): 2933–38. http://dx.doi.org/10.1039/c7ay00712d.

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He, Chang, Zhanquan Zhang, Boqiang Li, and Shiping Tian. "The Pattern and Function of DNA Methylation in Fungal Plant Pathogens." Microorganisms 8, no. 2 (February 8, 2020): 227. http://dx.doi.org/10.3390/microorganisms8020227.

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To successfully infect plants and trigger disease, fungal plant pathogens use various strategies that are dependent on characteristics of their biology and genomes. Although pathogenic fungi are different from animals and plants in the genomic heritability, sequence feature, and epigenetic modification, an increasing number of phytopathogenic fungi have been demonstrated to share DNA methyltransferases (MTases) responsible for DNA methylation with animals and plants. Fungal plant pathogens predominantly possess four types of DNA MTase homologs, including DIM-2, DNMT1, DNMT5, and RID. Numerous studies have indicated that DNA methylation in phytopathogenic fungi mainly distributes in transposable elements (TEs), gene promoter regions, and the repetitive DNA sequences. As an important and heritable epigenetic modification, DNA methylation is associated with silencing of gene expression and transposon, and it is responsible for a wide range of biological phenomena in fungi. This review highlights the relevant reports and insights into the important roles of DNA methylation in the modulation of development, pathogenicity, and secondary metabolism of fungal plant pathogens. Recent evidences prove that there are massive links between DNA and histone methylation in fungi, and they commonly regulate fungal development and mycotoxin biosynthesis.

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Burenina, O. Yu, E. A. Fedotova, A. Yu Ryazanova, A. S. Protsenko, M. V. Zakharova, A. S. Karyagina, A. S. Solonin, T. S. Oretskaya, and E. A. Kubareva. "Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction–Modification System." Acta Naturae 5, no. 2 (June 15, 2013): 70–80. http://dx.doi.org/10.32607/20758251-2013-5-2-70-80.

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Transcription regulation in bacterial restrictionmodification (RM) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI RM system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.

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Xue, Qingwang, Lei Wang, and Wei Jiang. "Label-free molecular beacon-based quadratic isothermal exponential amplification: a simple and sensitive one-pot method to detect DNA methyltransferase activity." Chemical Communications 51, no. 70 (2015): 13538–41. http://dx.doi.org/10.1039/c5cc05410a.

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Wu, Jiqin, Guoliang Lu, Bo Zhang, and Peng Gong. "Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation." Journal of Virology 89, no. 1 (October 15, 2014): 249–61. http://dx.doi.org/10.1128/jvi.02085-14.

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ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

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Meng, Bowen, Naomi Epp, Winsen Wijaya, Jan Mrázek, and Timothy R. Hoover. "Methylation Motifs in Promoter Sequences May Contribute to the Maintenance of a Conserved m5C Methyltransferase in Helicobacter pylori." Microorganisms 9, no. 12 (November 30, 2021): 2474. http://dx.doi.org/10.3390/microorganisms9122474.

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DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (m5C) MTase that converts GCGC motifs to Gm5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains.

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Birkholz, Nils, Simon A. Jackson, Robert D. Fagerlund, and Peter C. Fineran. "A mobile restriction–modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease." Nucleic Acids Research 50, no. 6 (March 14, 2022): 3348–61. http://dx.doi.org/10.1093/nar/gkac147.

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Abstract Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction–modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

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Khrabrova, Loiko, Tolkacheva, Cherepanova, Zvereva, Kirsanova, and Gromova. "Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia." Biomolecules 10, no. 1 (December 18, 2019): 8. http://dx.doi.org/10.3390/biom10010008.

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In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3–15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

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45

Christensen, Lisa Lystbæk, and Jytte Josephsen. "The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene." Journal of Bacteriology 186, no. 2 (January 15, 2004): 287–95. http://dx.doi.org/10.1128/jb.186.2.287-295.2004.

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ABSTRACT The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P llaDIIM ) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P llaDIIR ) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P llaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.

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46

Skoglund, Anna, Britta Björkholm, Christina Nilsson, Anders F. Andersson, Cecilia Jernberg, Katja Schirwitz, Cristofer Enroth, Margareta Krabbe, and Lars Engstrand. "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori." Journal of Bacteriology 189, no. 24 (October 5, 2007): 8914–21. http://dx.doi.org/10.1128/jb.00108-07.

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ABSTRACT A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

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47

Korzun, V., H. J. Balzer, A. Balzer, H. Bäumlein, and A. Börner. "Chromosomal location of three wheat sequences with homology to pollen allergen encoding, DNA replication regulating, and DNA (cytosine-5)-methyltransferase genes in wheat and rye." Genome 39, no. 6 (December 1, 1996): 1213–15. http://dx.doi.org/10.1139/g96-154.

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Three wheat sequences, shown to be homologous to pollen allergen encoding, DNA replication regulating, and DNA (cytosine-5)-methyltransferase genes were localized on chromosomes using nullisomic–tetrasomic wheat ('Chinese Spring') and wheat–rye ('Chinese Spring'/'Imperial') addition lines. Whereas the loci for the pollen allergen encoding sequence (Tri a III) were shown to be located on homoeologous group 4, the DNA replication regulating (Rep) and DNA (cytosine-5)-methyltransferase (Mtase) genes were located to homoeologous groups 1 and 7, respectively, of Triticeae. Chromosomal rearrangements in wheat and rye relative to each other are discussed. Key words : chromosomal location, known function probes, RFLP, rye, wheat.

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48

Park, Suk-Youl, Hyun-Ju Lee, Jung-Mi Song, Jiali Sun, Hyo-Jeong Hwang, Kosuke Nishi, and Jeong-Sun Kim. "Structural characterization of a modification subunit of a putative type I restriction enzyme fromVibrio vulnificusYJ016." Acta Crystallographica Section D Biological Crystallography 68, no. 11 (October 18, 2012): 1570–77. http://dx.doi.org/10.1107/s0907444912038826.

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In multifunctional type I restriction enzymes, active methyltransferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit fromVibrio vulnificusYJ016 (vvHsdM) was elucidated at a resolution of 1.80 Å. A cofactor-binding site forS-adenosyl-L-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an α/β-fold, in which a number of residues are conserved, including the GxGG and (N/D)PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase ofThermus aquaticusand other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group.

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49

Jois, Prashanth S., Nagaraj Madhu, and Desirazu N. Rao. "Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis." Biochemical Journal 410, no. 3 (February 27, 2008): 543–53. http://dx.doi.org/10.1042/bj20070900.

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Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5′-CAGCAG-3′. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

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50

Wang, Cong, Zhihua Guo, Ruifen Tian, Keying Zhang, Hongyan Wang, Fajun Li, Hongwei Shi, and Zhicheng Wang. "Ratiometric Electrochemical Biosensing of Methyltransferase Activity." Catalysts 12, no. 11 (November 4, 2022): 1362. http://dx.doi.org/10.3390/catal12111362.

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In this work, a novel ratiometric electrochemical readout platform was proposed and developed for the fast and flexible analysis of M.SssI methyltransferase (MTase) activity. In this platform, two hairpin DNAs (H1 and H2) were designed. H1 contains the palindromic sequence of 5′-CCGG-3′ in its stem which could be methylated and hybridize with H2 labeled by methylene blue (MB) as one of the signal reporters on a gold electrode (GE) in the presence of M.SssI MTase. Additionally, a specific immunoreaction was introduced by conjugating an anti-5-methylcytosine antibody, a DNA CpG methylation recognition unit, with 1,3-ferrocenedicarboxylic acid (Fc) as the second signal reporter. The results showed that when the Fc tag approaches, the MB tag was far from the gold electrode surface, resulting in a decrease in the oxidation peak current of MB (IMB) and an increase in the oxidation peak current of Fc (IFc). The ratiometric electrochemical method above shows the linear range of detection was 0 U/mL 40 U/mL with a detection limit of 0.083 U/mL (the mean signal of blank measures þ3s).

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