Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-l-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events—the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems

A directed evolution design of a GCG-specific DNA hemimethylase

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases

Investigations into metabolism, transport and function of sulfonated steroids in the porcine testicular-epididymal compartment

Sulfonated steroids have been traditionally regarded as inactive metabolites destined for excretion, as they are incapable of binding to classical nuclear steroid receptors. However, by the enzyme steroid sulfatase (STS) they may be converted into free steroids, which may be biologically active directly or after a few additional enzymatic reactions. Thus, as sulfonated steroids commonly circulate at relatively high concentrations, they may form an important pool of precursors for the local (intra-tissue) production of active free steroids. This so-called sulfatase pathway has received increased attention over recent years especially with respect to estrogen metabolism in human hormone-dependent breast cancer, where the intratumoral estrogen production from sulfonated precursors obviously has a much higher capacity in comparison to the de novo synthesis via free steroids. This study is composed of two parts of which the first one addresses the secretory patterns of free and sulfonated steroids in vivo, whereas in the second part the expression of STS and of the steroid sulfotransferases SULT1E1 (estrogen specific) and SULT2B1 (specific for beta-hydroxysteroids) was characterized in the testis and in different segments of the epididymis. Other subjects of the second part of this study were hydrolysis of steroid sulfates and the sulfonation of estrone (E1), dehydroepiandrosterone (DHEA) and pregnenolone (P5) in the tissues investigated. Concentrations of androstenedione, testosterone, pregnenolone sulfate (P5S), dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate (E1S)and 17beta-estradiol-3-sulfate were performed in the Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen (head: Prof. Dr. S. Wudy) applying liquid chromatography tandem mass spectrometry (LC-MS-MS). Moreover, 17beta-estradiol (E2) and E1 were measured by inhouse radioimmunoassays to cope with the low concentrations of free estrogens in boars. In order to get new information on the sulfonation of free steroids and the hydrolysis of steroid sulfates in the porcine testicular-epididymal compartment, subcellular fractions were prepared from tissue samples collected from the testis and from defined sites of the epididymis (EH1, EH2: proximal/distal part of epididymal head; EB1-4: epididymal body, from proximal to distal; ET1, ET2: proximal/distal part of epididymal tail) using differential centrifugation. STS and steroid sulfotransferase activities were measured based on the differential distribution of free and sulfonated steroids between an aqueous phase and an organic solvent, tert butyl-methylether. The immunostaining results were shown that SULTs 1E1 and 2B1, immunostaining was especially prominent in superficial epithelial protrusions. Sporadic staining of weaker intensity was also found in the muscular layer and in the vascular endothelium. With WB, a specific band of the expected molecular size (approx. 61 kDa) was found in the testis and all segments of the epididymis. These results show that STS is widely expressed in the porcine testicular-epididymal compartment, indicating a high potential for sulfatase pathways especially in Leydig cells and the epithelial cells of the rete testis and epididymis. The co-expression of STS with SULTs 1E1 and 2B1 in the epididymal epithelium and especially their colocalization in superficial protrusions are very intriguing. In the epididymal duct, apocrine secretion has been described to give rise to the formation of epididymosomes, small vesicles which are considered as vehicles for the transfer of certain molecules to the maturing sperm cells. Other intriguing findings are the virtual absence of a sulfonation of E1, DHEA and P5 in testicular cytosols as well as the absent or questionable detection of SULTs 1E1 and 2B1 in light of the high efflux of various steroid sulfates from the testis. A plausible explanation could be a significant use of sulfonated steroids as precursors/intermediates in porcine testicular steroidogenesis starting from cholesterol sulfate. The concept of a “sulfate pathway” of steroidogenesis would not only provide an explanation for the production of high amounts of steroid sulfates in the virtual absence of relevant steroid sulfotransferase activities but also for the high STS expression in Leydig cells. According to this concept, STS could play a crucial role in the control of the substrate flow through the steroidogenic enzyme cascade by mediating the transition of sulfonated precursors into the pool of free steroids, with the exact subcellular localization being of importance for the step of the enzyme cascade at which this transition(s) may occur. Thus, in order to corroborate this concept investigations into the utilization of sulfonated substrates by steroidogenic enzymes and on the subcellular localization of STS are necessary

Using shotgun sequence data to find active restriction enzyme genes

Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus

Selective recognition of pyrimidine–pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes

Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro

Novel non-specific DNA adenine methyltransferases

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments