Nucleoside-Driven Specificity of DNA Methyltransferase**

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l -methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l -methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.     

Targeted Mutagenesis Results in an Activation of DNA Methyltransferase 1 and Confirms an Autoinhibitory Role of its RFTS Domain

The N‐terminal regulatory part of DNA methyltransferase 1 (Dnmt1) contains a replication foci targeting sequence (RFTS) domain, which is involved in the recruitment of Dnmt1 to replication forks. The RFTS domain has been observed in a crystal structure to bind to the catalytic domain of the enzyme and block its catalytic centre. Removal of the RFTS domain led to activation of Dnmt1, thus suggesting an autoinhibitory role of this domain. Here, we destabilised the interaction of the RFTS domain with the catalytic domain by site‐directed mutagenesis and purified the corresponding Dnmt1 variants. Our data show that these mutations resulted in an up to fourfold increase in Dnmt1 methylation activity in vitro. Activation of Dnmt1 was not accompanied by a change in its preference for methylation of hemimethylated CpG sites. We also show that the Dnmt1 E572R/D575R variant has a higher DNA methylation activity in human cells after transfection into HCT116 cells, which are hypomorphic for Dnmt1. Our findings strongly support the autoinhibitory role of the RFTS domain, and indicate that it contributes to the regulation of Dnmt1 activity in cells.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Molecular drivers of base flipping during sequence-specific DNA methylation

One step at a time: Substrates containing nucleotide analogues lacking sequence-specific contacts to the C5 methyltransferase M.HhaI were used to probe the role of individual interactions in effecting conformational transitions during base flipping. A segregation of duties, that is, specific recognition and chemomechanical force for base flipping and active site assembly, within the enzyme is confirmed.     

Sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA)

A new concept for sequence-specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S-adenosyl-L-methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase-catalyzed sequence-specific coupling with DNA. Sequence-specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.     

Kaiso Regulates DNA Methylation Homeostasis

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.     

A selenium-based click AdoMet analogue for versatile substrate labeling with wild-type protein methyltransferases

Protein methylation is catalyzed by S-adenosyl-L-methionine-dependent protein methyltransferases (MTases), and this posttranslational modification serves diverse cellular functions. Some MTases seem to exhibit broad substrate specificities and comprehensive methods for target profiling are needed. Here we report the synthesis of a new AdoMet analogue for enzymatic transfer of a small propargyl group and labeling of modified proteins through copper-catalyzed azide-alkyne cycloaddition (CuAAC). Replacement of sulfur by selenium strongly enhanced the stability of the progargylic cofactor, leading, in combination with better activation by the selenonium center, to higher enzymatic reactivity. A broad spectrum of wild-type protein MTases acting on lysine, arginine, and glutamine residues accept this cofactor and modified substrates can be efficiently labeled by CuAAC click chemistry.     

Cofactor Regeneration of NAD+ from NADH: Novel Water‐Forming NADH Oxidases

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD⁺ can be considered solved, the inverse problem of regenerating NAD⁺ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD⁺ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O₂ either to undesirable H₂O₂ or to innocuous H₂O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O₂ to H₂O.     

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.     

Complex formation between plasmid DNA and self-aggregates of deoxycholic acid-modified chitosan

The use of non-viral delivery systems has numerous advantages in the area of gene therapy as this approach could circumvent the safety issues that may occur when using viral vectors. Hydrophobically modified chitosan containing five deoxycholic acid groups per 100 anhydroglucose units was synthesized to prepare self-aggregated nanoparticles in aqueous media and utilized to form complexes with plasmid DNA. Formation and various characteristics of DNA/chitosan self-aggregate complexes were investigated by electrophoresis, zeta potential measurement, and dynamic light scattering method. It was hypothesized that complex formation between DNA and chitosan self-aggregates can be strongly dependent on the charge ratio, pH, and incubation time. Migration of DNA on an agarose gel was completely retarded over a charge ratio (+/−) of 4/1 at pH 7.2, and the mean diameter of the complexes was determined to be 350 nm with unimodal size distribution (+/−=4/1; pH 7.2; incubation time, 30 min). The complex formation behavior was also regulated by pH of the media. The incubation time was unlikely to be the critical factor affecting the complex formation behavior due to the instant electrostatic interactions between negatively charged DNA and positively charged chitosan self-aggregates. This approach to controlling the characteristics of DNA/self-aggregate complexes could be critical in the design and tailoring of novel gene delivery systems using non-viral vectors.     

Kinetic and transport effects on enzymatic biocatalysis resulting from the PEGylation of cofactors

The utilization of cofactor‐dependent redox enzymes in bioprocess technologies requires low cost cofactor regeneration methods. PEGylated NAD(H) (PEG‐NAD(H)) has been utilized in enzyme membrane reactors as a means to recover the cofactor; however, there is a lack of understanding of the effect of PEGylation on enzymatic activity, especially on the relationship between biocatalysis and transport phenomena. To explore this further, two redox enzymes (formate dehydrogenase (FDH) from Saccharomyces cerevisiae and NAD(H)‐dependent d ‐lactate dehydrogenase (nLDH) from Escherichia coli) have been chosen and the kinetic effects caused by cofactor modifications (with PEG of three different chain lengths) have been investigated. The PEGylation did not impact the cofactor dissociation constants and mass transfer was not the rate‐limiting step in biocatalysis for either enzyme. However, the PEG chain length had different impacts on the formation of enzyme/cofactor and/or enzyme/cofactor/substrate ternary complexes for the enzymes. © 2017 American Institute of Chemical Engineers AIChE J, 63: 12–17, 2018     

Expression Patterns of DNA Methylation and Demethylation Genes during Plant Development and in Response to Phytohormones

DNA methylation and demethylation precisely and effectively modulate gene expression during plant growth and development and in response to stress. However, expression profiles of genes involved in DNA methylation and demethylation during plant development and their responses to phytohormone treatments remain largely unknown. We characterized the spatiotemporal expression patterns of genes involved in de novo methylation, methyl maintenance, and active demethylation in roots, shoots, and reproductive organs using β-glucuronidase (GUS) reporter lines. Promoters of DNA demethylases were generally more highly active at the mature root tissues, whereas the promoters of genes involved in DNA methylation were more highly active at fast-growing root tissues. The promoter activity also implies that methylation status in shoot apex, leaf primordia, floral organs, and developing embryos is under tight equilibrium through the activity of genes involved in DNA methylation and demethylation. The promoter activity of DNA methylation and demethylation-related genes in response to various phytohormone treatments revealed that phytohormones can alter DNA methylation status in specific and redundant ways. Overall, our results illustrate that DNA methylation and demethylation pathways act synergistically and antagonistically in various tissues and in response to phytohormone treatments and point to the existence of hormone-linked methylome regulation mechanisms that may contribute to tissue differentiation and development.     

Metabolic labeling of DNA by purine analogues in vivo

DNA tracking: The 2'-deoxyadenosine analogue "EdA" efficiently labels newly synthesized DNA with alkyne groups in vivo without inhibiting cell cycle progression or embryonic development. This provides the first demonstration that a bio-orthogonal functional group can be metabolically incorporated into DNA through endogenous pathways by addition of a purine analogue to cells.     

DNA的定量分析方法进展

对近年来脱氧核糖核酸(DNA)的定量分析方法的进展进行了评述,列出了重要的反应体系及分析特征.     

A Mutation-Based Method for Pinpointing a DNA N6-Methyladenine Methyltransferase Modification Site at Single Base Resolution

DNA N⁶-methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N⁶-position of adenine within a specific DNA sequence to form N⁶-allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N⁶-cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.     

DNA mismatch-specific base flipping by a bisacridine macrocycle

Most, if not all, enzymes that chemically modify nucleobases in DNA flip their target base from the inside of the double helix into an extrahelical position. This energetically unfavorable conformation is partly stabilized by specific binding of the apparent abasic site being formed. Thus, DNA base-flipping enzymes, like DNA methyltransferases and DNA glycosylases, generally bind very strongly to DNA containing abasic sites or abasic-site analogues. The macrocyclic bisacridine BisA has previously been shown to bind abasic sites. Herein we demonstrate that it is able to specifically recognize DNA base mismatches and most likely induces base flipping. Specific binding of BisA to DNA mismatches was studied by thermal denaturation experiments by using short duplex oligodeoxynucleotides containing central TT, TC, or TG mismatches or a TA match. In the presence of the macrocycle a strong increase in the melting temperature of up to 7.1 degrees C was observed for the mismatch-containing duplexes, whereas the melting temperature of the fully matched duplex was unaffected. Furthermore, BisA binding induced an enhanced reactivity of the mispaired thymine residue in the DNA toward potassium permanganate oxidation. A comparable reactivity has previously been observed for a TT target base mismatch in the presence of DNA methyltransferase M.TaqI. This similarity to a known base-flipping enzyme suggests that insertion of BisA into the DNA helix displaces the mispaired thymine residue into an extrahelical position, where it should be more prone to chemical oxidation. Thus, DNA base flipping does not appear to be limited to DNA-modifying enzymes but it is likely to also be induced by a small synthetic molecule binding to a thermodynamically weakened site in DNA.