Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1

We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase 1 (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26-bp hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic read-out method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage that would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format, and trivial changes in the substrate sequence and endonuclease allow adaptation of the method to any bacterial or human DNA methyltransferase.     

The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.     

New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases

An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. As an example, we have studied methylation of DNA by the EcoRV DNA methyltransferase. The reaction progress curves measured with this assay are linear with respect to time. Methylation rates linearly increase with enzyme concentration. The rates are comparable to results obtained with the same enzyme using a different assay. The biotin-avidin assay is inexpensive, convenient, quantitative, fast and well suited to process many samples in parallel. The accuracy of the assay is high, allowing to reproduce results within +/- 10%. The assay is very sensitive as demonstrated by the detection of incorporation of 0.8 fmol methyl groups into the DNA. Under the experimental conditions, this corresponds to methylation of only 0.03% of all target sites of the substrate. Using this assay, the DNA methylation activity of some M.EcoRV variants could be detected that was not visible by other in vitro methylation assays.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

An easy, quantitative method for detection of endonuclease activity

An easy yet sensitive assay has been developed for the detection of endonuclease activities. The method involves the use of agarose gel electrophoresis to resolve intact homogeneous nucleic acid substrate from degradation products resulting from a small number of nucleolytic breaks. The assay is quantitative when a radioactively labeled nucleic acid is used as substrate, and it is as sensitive in the measurement of nuclease activity as is zone sedimentation in sucrose gradients. The assay can detect as few as 1.4 nicks, on the average, per substrate molecule. Its advantage over previous methods of analysis is the ease with which large numbers of samples can be handled while still retaining a high degree of sensitivity. The method is demonstrated with single-stranded DNA substrate, but it can be easily modified to detect endonuclease degradation of double-stranded DNA or degradation of RNA substrate.     

Mechanism of action of Escherichia coli endonuclease III

Endonuclease III isolated from Escherichia coli has been shown to have both N-glycosylase and apurinic/apyrimidinic (AP) endonuclease activities. A nicking assay was used to show that the enzyme exhibited a preference for form I DNA when DNA containing thymine glycol was used as a substrate. This preference was reduced or eliminated either when the DNA was relaxed or when the type of damage was altered to urea residues or AP sites. The combined N-glycosylase/AP endonuclease activity was at least 10-fold higher than the AP endonuclease activity alone when urea-containing DNA was used as a substrate as compared to AP DNA. When DNA containing thymine glycol was used as a substrate, the combined N-glycosylase/AP endonuclease activity was about 2-fold higher than the AP endonuclease activity. Yet, when DNA containing thymine glycol or urea was used as substrate, no apurinic sites remained. Furthermore, magnesium selectively inhibited endonuclease III activity when AP DNA was used as a substrate but had no effect when DNA containing either urea or thymine glycol was used as substrate. These data suggest that both the N-glycosylase and AP endonuclease activities of endonuclease III reside on the same molecule or are in very tight association and that these activities act in concert, with the N-glycosylase reaction preceding the AP endonuclease reaction.     

Methylated DNA labels for marking objects

We recently described a method for digitally labelling objects with DNA. Here we show that, using DNA methyltransferases to create polymorphic DNA templates, it is possible to significantly increase the number of labels that can be generated by this method. Nine double-stranded DNA templates of different length were methylated with either M.HaeIII or M.AluI methyltransferase, or both. Different mixtures of methylated and unmethylated versions of this template set were used to 'invisibly' label paper. The mixtures were eluted from the paper and the methylated status of the templates in each mixture successfully determined, and the labels read, by digestion with the complementary restriction endonuclease, followed by a polymerase chain reaction and agarose gel electrophoresis. One methylated DNA label was read after it had been left on paper for two months.     

Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase

Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K(m) and k(cat) values of the Dim-5 enzyme for the methylation of a 20 mer peptide to be 7.4 microM and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.     

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.     

Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.

A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

限制性核酸内切酶与DNA相互作用研究进展

蛋白质对ＤＮＡ识别的模体中,除了锌指结构、螺旋—转角—螺旋、亮氨酸拉链和β带外,近年来发现,Ⅱ型限制性内切酶与ＤＮＡ作用的模体有许多特别之处。通过对ＥｃｏＲＩ、ＢａｍＨＩ、ＥｃｏＲＶ等与ＤＮＡ复合物的空间构象、一级结构分析,发现酶分子存在催化性裂缝,并且氨基端形成臂结构包绕ＤＮＡ;同时ＤＮＡ发生构象变化、螺旋扭结。这些有趣的结构有利于酶对底物的特异性结合和催化作用。     

The Unique FauI Restriction–Modification System: Cloning and Protein Sequence Comparisons

The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction–modification system. The operon is unusual in structure and has the gene order control protein / DNA methyltransferase A / restriction endonuclease / DNA methyltransferase B, other than in the known analogs; the genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction–modification systems, and the restriction endonuclease shows low similarity to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

A novel assay for endonucleases acting at apurinic sites and its use in measuring AP endonuclease activity in repair-deficient mutants of Saccharomyces cerevisiae.

A quick and convenient assay for depurination and AP endonuclease activities has been developed. (The term 'AP endonuclease' refers to a nuclease that acts on apurinic and probably apyrimidinic sites on DNA.) It is based on the observation that different topological forms of DNA, such as open circular DNA and covalently closed circular DNA, bind different amounts of the fluorescent intercalator ethidium bromide, and can therefore be distinguished by their fluorescence. This assay has been used to measure AP endonuclease activity in 22 repair-deficient mutants of Saccharomyces cerevisiae. All 22 had normal or nearly normal AP endonuclease activity. The AP endonuclease activity was partially characterized.     

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based ‘High-Throughput Methyl Reading’ (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.     

Trapped in action: direct visualization of DNA methyltransferase activity in living cells

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.     

DNA甲基转移酶

DNA甲基化在基因调节和动物发育中起着重要作用。负责DNA甲基化作用的酶尔为DNA甲基转移酶（Dnmts）。到目前为止,在哺乳动物细胞中已经鉴定了三种DNA甲基转移酶基因家族,即Dnmt1、Dnmt2和Dnmt3。鉴定和研究DNA甲基转移酶对阐明DNA甲基化机制起着关键的作用。