Specificity of DNA triple helix formation analyzed by a FRET assay

BACKGROUND: 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. RESULTS: 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 × 10(5) and 2.3 × 10(6) M(-1). Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg(2+)ions whereas it is inhibited by K(+)ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 10(4) and 8.1 × 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. CONCLUSION: 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

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

DNA methyltransferase 1 (Dnmt1) has a central role in copying the pattern of DNA methylation after replication which is one manifestation of epigenetic inheritance. With oligonculeotide substrates we show that mouse Dnmt1 has a 30- to 40-fold preference for hemimethylated DNA that is almost lost after addition of fully methylated oligonucleotides. Using long hemimethylated DNA substrates that carry defined methylation patterns and bisulfite analysis of the methylation reaction products, we show a 15-fold preference for hemimethylated CG sites. Dnmt1 moves along the DNA in a random walk methylating hemimethylated substrates with high processivity (>50 sites are visited on average which corresponds to linear diffusion over 6000 bp). The frequency of skipping sites is very low (<0.3%) and there is no detectable flanking sequence preference. CGCTC sites tend to terminate the processive methylation of DNA by Dnmt1. Unmethylated DNA is modified non-processively with a preference for methylation at CCGG sites. We simulate the propagation of methylation patterns using a stochastic model with the specificity of Dnmt1 observed here and conclude that either methylation of several sites is required to propagate the methylation information over several cellular generations or additional epigenetic information must be used

BISMA - Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences

<p>Abstract</p> <p>Background</p> <p>Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution.</p> <p>Results</p> <p>The BISMA (Bisulfite Sequencing DNA Methylation Analysis) software for analysis of primary bisulfite sequencing data implements sequencing data extraction and enhanced data processing, quality controls, analysis and presentation of the methylation state. It uses an improved strategy for detection of clonal molecules and accurate CpG site detection and it supports for the first time analysis of repetitive sequences.</p> <p>Conclusions</p> <p>BISMA works highly automated but still provides the user full control over all steps of the analysis. The BISMA software is freely available as an online tool for academic purposes for the analysis of bisulfite sequencing data from both unique and repetitive sequences <url>http://biochem.jacobs-university.de/BDPC/BISMA/</url>.</p

The inhibition of the mammalian DNA methyltransferase 3a (Dnmt3a) by dietary black tea and coffee polyphenols

<p>Abstract</p> <p>Background</p> <p>Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean.</p> <p>Results</p> <p>We studied the inhibition of DNA methyltransferase 3a by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC₅₀values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance.</p> <p>Conclusions</p> <p>Since Dnnmt3a has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols.</p

Allosteric control of mammalian DNA methyltransferases - a new regulatory paradigm

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 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

Somatic cancer mutations in the MLL1 histone methyltransferase modulate its enzymatic activity and dependence on the WDR5/RBBP5/ASH2L complex

Somatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1–WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1–WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1–WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered

Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

<p>Abstract</p> <p>Background</p> <p>Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains.</p> <p>Results</p> <p>We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification.</p> <p>Conclusions</p> <p>We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.</p

Bisulfite sequencing Data Presentation and Compilation (BDPC) web server—a useful tool for DNA methylation analysis

During bisulfite genomic sequencing projects large amount of data are generated. The Bisulfite sequencing Data Presentation and Compilation (BDPC) web interface (http://biochem.jacobs-university.de/BDPC/) automatically analyzes bisulfite datasets prepared using the BiQ Analyzer. BDPC provides the following output: (i) MS-Excel compatible files compiling for each PCR product (a) the average methylation level, the number of clones analyzed and the percentage of CG sites analyzed (which is an indicator of data quality), (b) the methylation level observed at each CG site and (c) the methylation level of each clone. (ii) A methylation overview table compiling the methylation of all amplicons in all tissues. (iii) Publication grade figures in PNG format showing the methylation pattern for each PCR product embedded in an HMTL file summarizing the methylation data, the DNA sequence and some basic statistics. (iv) A summary file compiling the methylation pattern of different tissues, which is linked to the individual HTML result files, and can be directly used for presentation of the data in the internet. (v) A condensed file, containing all primary data in simplified format for further downstream data analysis and (vi) a custom track file for display of the results in the UCSC genome browser

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4–8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(γ-maleimidobutryloxy) succinimide ester to a TFO (5′-NH(2)-[CH(2)](6 or 12)-MPMPMPMPMPPPPPPT-3′, with M being 5-methyl-2′-deoxycytidine and P being 5-[1-propynyl]-2′-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO (‘addressed’ site: 5′-TTTTTTTCTCTCTCTCN(∼10)CAGCTG-3′), leaving ‘unaddressed’ PvuII sites intact. The preference for cleavage of an ‘addressed’ compared to an ‘unaddressed’ site is >1000-fold, if the cleavage reaction is initiated by addition of Mg(2+) ions after preincubation of scPvuII-TFO and substrate in the absence of Mg(2+) ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO

Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation

Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation. Using an mf-Lon protease degradable CcrM variant enables reversible switching. Epigenetic memory systems have numerous potential applications in synthetic biology, including life biosensors, death switches or induction systems for industrial protein production. The large variety of bacterial DNA methyltransferases potentially allows for massive multiplexing of signal storage and logical operations depending on more than one input signal