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Circularly permuted variants of two CG-specific prokaryotic DNA methyltransferases

Pál Albert, Bence Varga, Nikolett Zsibrita, Antal Kiss, Albert Jeltsch
2018 PLoS ONE  
The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.MpeI and M.SssI share the specificity of eukaryotic C5-MTases (5'-CG), and can be useful research tools in the study of eukaryotic DNA methylation and epigenetic regulation. In an effort to improve the stability and solubility of complementing fragments of the two MTases, genes encoding circularly permuted (CP) variants of M.MpeI and M.SssI were created, and cloned in a plasmid vector downstream of an
more » ... cible promoter. MTase activity of the CP variants was tested by digestion of the plasmids with methylation-sensitive restriction enzymes. Eleven of the fourteen M.MpeI permutants and six of the seven M.SssI permutants had detectable MTase activity as indicated by the full or partial protection of the plasmid carrying the cpMTase gene. Permutants cp62M.MpeI and cp58M.SssI, in which the new N-termini are located between conserved motifs II and III, had by far the highest activity. The activity of cp62M.MpeI was comparable to the activity of wild-type M.MpeI. Based on the location of the split sites, the permutants possessing MTase activity can be classified in ten types. Although most permutation sites were designed to fall outside of conserved motifs, and the MTase activity of the permutants measured in cell extracts was in most cases substantially lower than that of the wild-type enzyme, the high proportion of circular permutation topologies compatible with MTase activity is remarkable, and is a new evidence for the structural plasticity of C5-MTases. A computer search of the REBASE database identified putative C5-MTases with CP arrangement. Interestingly, all natural circularly permuted C5-MTases appear to represent only one of the ten types of permutation topology created in this work.
doi:10.1371/journal.pone.0197232 pmid:29746549 pmcid:PMC5944983 fatcat:raebn4jbsrfzjobjrwsvdksglm

Quality of histone modification antibodies undermines chromatin biology research

Goran Kungulovski, Albert Jeltsch
2015 F1000Research  
Competing Interests: Author Response 24 Nov 2015 , Uni Stuttgart, Germany Albert Jeltsch Scott B.  ...  Competing Interests: Author Response 12 Nov 2015 , Uni Stuttgart, Germany Albert Jeltsch "I agree with the Kungulovski and Jeltsch that increased accountability needs to be demanded from companies who  ... 
doi:10.12688/f1000research.7265.2 pmid:26834995 pmcid:PMC4706057 fatcat:swb3iqc5aneshfnpzn4bsvxdv4

Continuous enzymatic assay for histone lysine methyltransferases

Philipp Rathert, Xiaodong Cheng, Albert Jeltsch
2007 BioTechniques  
Since it yields quantitative results, it can be used for Continuous enzymatic assay for histone lysine methyltransferases Philipp Rathert 1 , Xiaodong Cheng 2 , and Albert Jeltsch We describe a continuous  ... 
doi:10.2144/000112623 pmid:18072589 pmcid:PMC2703000 fatcat:ob5kqe7avnbi3avr7tsnjgmbku

German Human Methylome Project Started

Albert Jeltsch, Jörn Walter, Richard Reinhardt, Matthias Platzer
2006 Cancer Research  
Albert Jeltsch Richard ReinhardtMax Planck Institute for Molecular Genetics, Ihnestrasse, Berlin-Dahlem, Germany Matthias Platzer Leibniz Institute for Age Research-Fritz Lipmann Institute, Beutenbergstr  ... 
doi:10.1158/0008-5472.can-06-1071 pmid:16849590 fatcat:b7t6jold7fecdmfpw4fmivvwxu

Quality of histone modification antibodies undermines chromatin biology research

Goran Kungulovski, Albert Jeltsch
2015 F1000Research  
Author Response 12 Nov 2015 , Uni Stuttgart, Germany Albert Jeltsch "I agree with the Kungulovski and Jeltsch that increased accountability needs to be demanded from companies who sell histone PTM antibodies  ...  Author Response 12 Nov 2015 , Uni Stuttgart, Germany Albert Jeltsch "Paragraph 2 should also indicate that antibodies purportedly recognizing PTMs at specific sites, need to have their recognition specificity  ... 
doi:10.12688/f1000research.7265.1 pmid:26834995 pmcid:PMC4706057 fatcat:hwew7y6jwzfcrgcqpb2fyjslvi

DNA fromAspergillus flavuscontains 5-methylcytosine

Humaira Gowher, Kenneth C. Ehrlich, Albert Jeltsch
2001 FEMS Microbiology Letters  
DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of
more » ... nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400. ß
doi:10.1111/j.1574-6968.2001.tb10939.x pmid:11728730 fatcat:z54ayostxvbblhf5oyxfqlgrri

DNMT1-associated DNA methylation changes in cancer

Pavel Bashtrykov, Albert Jeltsch
2014 Cell Cycle  
Cancer arises as a consequence of somatic mutations and clonal amplification of tumor cells. However, it has been known since many years that cancer cells also accumulate a multitude of epigenetic alterations including aberrant DNA methylation. 1 Interestingly, two converse trends of DNA methylation changes were observed in many tumors. On the one hand, promoters of tumor suppressor genes are often hypermethylated leading to the silencing of the genes. On the other hand, a global DNA
more » ... tion including heterochromatic repetitive elements is observed which leads to genomic instability. In a recent publication in Cell Cycle, Wu et al. 2 for the first time present a mechanism that could explain both of these inverse changes by a mutation in DNMT1, an important DNA methyltransferase in human cells. In mammals, DNA methylation mainly takes place at CpG sites, where methylation occurs on both DNA strands and it cycles between full methylation and hemimethylation after DNA replication. DNMT1 has a preference for the methylation of hemimethylated CpG sites and it was initially believed to purely function as maintenance methyltransferase, which copies DNA methylation patterns after replication. However, it became clear that DNA methylation is dynamically regulated and the control of DNA methyltransferases including DNMT1 plays a central role in the generation and maintenance of DNA methylation patterns. 3 DNMT1 is a large multidomain protein. Its C-terminal catalytic domain is under tight allosteric control by several N-terminal domains, including the RFTS domain (RFTD), which has 3 roles in the regulation of DNMT1: 2,4-7 First, it blocks the catalytic pocket and by this it reduces the catalytic activity of DNMT1. Second, it interacts with other proteins including UHRF1, which can target DNMT1 to replication foci (via its interaction with hemimethylated DNA), but also to heterochromatin (via its interaction with H3K9 methylated histones). Third, interaction with UHRF1 can relieve the RFTD mediated allosteric repression of DNMT1 activity. Wu et al. now show that deletion of the RFTS domain leads to converse changes of DNA methylation which mimic the alterations observed in cancer cells (Fig. 1) . One the one hand, the activity of DNMT1 is increased leading to DNA hypermethylation at sites, where DNMT1 is targeted in an RFTD independent manner, for example at promoter region of tumor suppressor genes. One the other hand, deletion of RFTD disrupts the interaction of DNMT1 and UHRF1 leading to the loss of heterochromatic localization DNMT1 and hypomethylation of heterochromatic SAT2 elements. It is conceivable to speculate that somatic DNMT1 mutations in cancer cells which disrupt folding of the RFTS domain could have similar effects. Therefore, the paper by Wu et al. is an important contribution to our understanding of the causes of the complex DNA methylation changes observed in cancer cells.
doi:10.4161/15384101.2014.989963 pmid:25483051 pmcid:PMC4353171 fatcat:vjnmlu3ptbhz3nlqfmlsa2r4em

Special Issue "Structure, Activity, and Function of Protein Methyltransferases"

Arunkumar Dhayalan, Albert Jeltsch
2022 Life  
Post-translational modifications (PTMs) largely expand the functional diversity of the proteome [...]
doi:10.3390/life12030405 pmid:35330156 pmcid:PMC8948979 fatcat:mlyd5qdlynerjlwhn5are7gbjq

Enzymes that keep DNA under control

Alfred Pingoud, Albert Jeltsch, Anthony Maxwell, David Sherratt
2001 EMBO Reports  
Jeltsch (Giessen, Germany) dealt with the Dnmt1, Dnmt3a and Dnmt3b enzymes.  ...  Rao (Bangalore, India), Gumport and Jeltsch discussed the molecular enzymology of four prokaryotic MTases (M.HhaI, M.EcoP15, M.RsrI and M.EcoRV).  ... 
doi:10.1093/embo-reports/kve073 pmcid:PMC1083870 fatcat:avy7aehxefberaj3q5zjsth5g4

The Application of Next Generation Sequencing in DNA Methylation Analysis

Yingying Zhang, Albert Jeltsch
2010 Genes  
DNA methylation is a major form of epigenetic modification and plays essential roles in physiology and disease processes. In the human genome, about 80% of cytosines in the 56 million CpG sites are methylated to 5-methylcytosines. The methylation pattern of DNA is highly variable among cells types and developmental stages and influenced by disease processes and genetic factors, which brings considerable theoretical and technological challenges for its comprehensive mapping. Recently various
more » ... -throughput approaches based on bisulfite conversion combined with next generation sequencing have been developed and applied for the genome wide analysis of DNA methylation. These methods provide single base pair resolution, quantitative DNA methylation data with genome wide coverage. We review these methods here and discuss some technical points of special interest like the sequence depth necessary to reach conclusions, the identification of clonal DNA amplification after bisulfite conversion and the detection of non-CpG methylation. Future application of these methods will greatly facilitate the profiling of the DNA methylation in the genomes of different species, individuals and cell types under healthy and disease states.
doi:10.3390/genes1010085 pmid:24710012 pmcid:PMC3960863 fatcat:lf5torm5drbgzczvdje74o64by

Specificity of DNA triple helix formation analyzed by a FRET assay

Sabine Reither, Albert Jeltsch
2002 BMC Biochemistry  
A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis. We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 x 10(5) and 2.3 x 10(6) M(-1). Our
more » ... confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ ions whereas it is inhibited by K+ ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 x 10(4) and 8.1 x 10(4) min(-1) M(-1). Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation. We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.
pmid:12323077 pmcid:PMC128820 fatcat:uka2oawkezeetgxtdazp7wuudq

On the Substrate Specificity of DNA Methyltransferases

Albert Jeltsch, Frauke Christ, Mehrnaz Fatemi, Markus Roth
1999 Journal of Biological Chemistry  
Jeltsch, manuscript in preparation. nine-N 6 MTases, the nature of the methylated base must be identified.  ... 
doi:10.1074/jbc.274.28.19538 pmid:10391886 fatcat:tfh4tc3ofjci3jmcrc62jahwqq

Allosteric control of mammalian DNA methyltransferases – a new regulatory paradigm

Albert Jeltsch, Renata Z. Jurkowska
2016 Nucleic Acids Research  
In mammals, DNA methylation is introduced by the DNMT1, DNMT3A and DNMT3B methyltransferases, which are all large multi-domain proteins containing a catalytic C-terminal domain and an N-terminal part with regulatory functions. Recently, two novel regulatory principles of DNMTs were uncovered. It was shown that their catalytic activity is under allosteric control of N-terminal domains with autoinhibitory function, the RFT and CXXC domains in DNMT1 and the ADD domain in DNMT3. Moreover, targeting
more » ... and activity of DNMTs were found to be regulated in a concerted manner by interactors and posttranslational modifications (PTMs). In this review, we describe the structures and domain composition of the DNMT1 and DNMT3 enzymes, their DNA binding, catalytic mechanism, multimerization and the processes controlling their stability in cells with a focus on their regulation and chromatin targeting by PTMs, interactors and chromatin modifications. We propose that the allosteric regulation of DNMTs by autoinhibitory domains acts as a general switch for the modulation of the function of DNMTs, providing numerous possibilities for interacting proteins, nucleic acids or PTMs to regulate DNMT activity and targeting. The combined regulation of DNMT targeting and catalytic activity contributes to the precise spatiotemporal control of DNMT function and genome methylation in cells.
doi:10.1093/nar/gkw723 pmid:27521372 pmcid:PMC5062992 fatcat:n7zfoprcgzdubpd7xk4xlda3i4

MOESM1 of Application of recombinant TAF3 PHD domain instead of anti-H3K4me3 antibody

Goran Kungulovski, Rebekka Mauser, Richard Reinhardt, Albert Jeltsch
2016 Figshare  
Additional file 1. Supplementary figures and table.
doi:10.6084/m9.figshare.c.3622751_d1.v1 fatcat:pn4thg4vnre2rfbrf4teeyn5mi

Multiplexed Sequence Encoding: A Framework for DNA Communication

Bijan Zakeri, Peter A. Carr, Timothy K. Lu, Albert Jeltsch
2016 PLoS ONE  
Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified
more » ... pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. Introduction Communication has many faces. While the general objective of transferring information between different parties remains constant, the medium for information transfer continues to evolve. In nature, DNA has been used for billions of years as the chemical of choice for transferring information across cells, species, and generations [1]. Now, advances in biotechnology are enabling the use of DNA for the transfer and storage of non-biological information [2] [3] [4] [5] [6] [7] [8] [9] . Rapid advances in digital technologies over the past decades have enabled efficient and facile communication regardless of whether our messages are short tweets of 140 characters or long communiqués of thousands of pages. However, our ever-increasing reliance on digital PLOS ONE | technologies may make it worthwhile to explore new methods of information storage and transfer with alternative characteristics. Key attributes of DNA including high-density data storage, static and stable data maintenance, efficient reproducibility, and the lack of technological obsolescence, mean that DNA has distinct advantages over current magnetic and optical data storage platforms [1], thus warranting further exploration into DNA-specific writing and reading technologies. With synthesis and sequencing speeds rising, and costs rapidly declining [10, 11] , DNA is an intriguing option for the transfer and storage of digital information [1]. DNA molecules have been used for hiding messages [2] and storing digital data [7] [8] [9] . In these studies, an encoding algorithm was used to convert digital data into nucleotide sequences that were then written, transferred, and read using DNA as the storage medium. The encoding algorithms were also programmed to reduce homopolymeric stretches as bits and trits were converted to bases. These methods allow users without prior biological or computer programming skills to encode any computer file into DNA sequences that can then be synthesized by commercial vendors. Therefore, we identified these three pivotal points of a communication-data encoding, data transfer & data extraction-to develop new methods for DNA-based communications (Fig 1a) . To illustrate, if Alice sends a message (m) to Bob, she would first write-encode and synthesize-the information in DNA molecules and send it to Bob who would then read-sequence and decode-the message (m). However, during the transfer of m between Alice and Bob, Eve could intercept the communication and read m. To protect the information stored in m, DNA-specific cryptography and steganography methods may be implemented [12] [13] [14] [15] , akin to conventional digital data transfer that incorporate encryption algorithms such as AES, RSA, Twofish, and others. However, in these early days of DNA data storage and communication, we believe it to be also useful to explore different methods of data encoding, data transfer, and data extraction to find additional opportunities afforded by DNA compared to conventional magnetic and optical platforms. Here we present a new framework for the communication of short messages in DNA that explores the fragmentation of a message across multiple distinct DNA molecules (Fig 1b) . To encode a message (m) in DNA, we used an encoding key (k) to convert plaintext into bases, while at the same time reducing the occurrence of homopolymers. k was designed as a substitution cipher that assigned 'codons' to characters based on the frequency of occurrence of characters in English text. Additionally, decoy messages (d) were also encoded using k and incorporated within a communication. To decode the information, a combination key is required to discern the message m from the decoy information d, where subsequently k can be used to decode m. To transfer data, we established a secret-sharing system where m can be fragmented across a mixture of different DNA molecules, requiring Eve to physically intercept and interrogate multiple separate data transmission lines to gain access to m. To facilitate data extraction, we investigated a new method that allows for the multiplexed sequencing of multiple DNA molecules with a common primer, where regions within distinct DNA molecules that have matching information can be identified from a single sequencing reaction via chromatogram patterning. Materials and Methods Plasmids Constructs were cloned using standard molecular biology techniques. KOD Hot Start DNA Polymerase (VWR) was used to PCR amplify a p15A origin and a chloramphenicol resistance gene, which were then fused to gBlocks from IDT (Table 1) using Gibson assembly. Random DNA sequences were generated at http://www.bioinformatics.org/sms2/random_dna.html. Constructs were sequence verified by Genewiz Inc. (Cambridge, MA). are no other declarations relating to employment, consultancy, patents, products in development or modified products, etc. combinations tuned and co-sequenced, DNA sequence of embedded messages, and close-ups of the chromatogram patterns produced are shown for the WWII communication including: (a) the combination key, (b), (c), (d), the desired message, and (e), (f), (g), the decoy message. Space1 was used for all odd numbered strands (n1, n3, n5) and space2 was used for all even numbered strands (n2, n4, n6) to demarcate words. Space1/2 codons are shown in red.
doi:10.1371/journal.pone.0152774 pmid:27050646 pmcid:PMC4822886 fatcat:wdhgsvyyprbu3ku36x42y5hmzq
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