Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin‐binding aptamer

The preferred residence sites and the conformation of DNA‐bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective ¹³C‐edited and selective total correlation spectroscopy‐edited one‐dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular ¹H nuclear Overhauser effect (NOE) connectivities in ¹³C‐labelled spermine bound to the thrombin‐binding aptamer. The results provide evidence that the aptamer‐bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6–C7 bonds, which results in either a curved or S‐shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex‐binding drugs. Copyright © 2013 John Wiley & Sons, Ltd.     

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles^1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues^1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)^3-7. Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde^8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies^9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C₄ photosynthesis in maize^5,11 and systemic immunity in Arabidopsis³.     

Effect of 5′-methylthioadenosine on induction of murine erythroleukemia cell differentiation

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of proliferation and differentiation. The present study has monitored the effects of 5′-methylthioadenosine, the metabolic product of spermidine and spermine synthesis, on the appearance of a differentiated murine erythroleukemia cell phenotype. The results demonstrate that increasing concentrations of 5′-methylthioadenosine (1 × 10^?6 to 5 × 10^?4M) progressively inhibit murine erythroleukemia cell heme synthesis and hemoglobin production. The results also demonstrate that this inhibition of differentiation is not related to depletion of intracellular spermidine or cytostasis. Since 5′-methylthioadenosine is also a known inhibitor of DNA methylation, this naturally occurring nucleoside may be an intermediate involved in both murine erythroleukemia cell proliferation and differentiation.     

Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein–DNA system

N¹-meA and N³-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S_N2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein–DNA crystallization system. The crystal structure of N¹-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N¹-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N¹-meA, N³-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N⁶-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N¹-meA or N³-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.     

Polyamine acetylations in normal and neoplastic growth processes

Summary The expression patterns of cytosolic and nuclear polyamine acetyltransferases were studied in normal and neoplastic growth processesin vivo andin vitro to evidentiate the roles played by these enzymes in cell proliferation. In regenerating liver, cytosolic spermidine/spermine N¹-acetyltransferase showed similar augments of mRNA level and enzymatic activity during the prereplicative period (4–8 h), whereas spermidine N⁸-acetyltransferase activity increased later (24 h) when DNA synthesis was maximally enhanced. In fibroblasts continuously dividing, the messenger for spermidine/spermine N¹-acetyltransferase rapidly accumulated after serum-stimulation. In cultured Morris hepatoma cells stimulated to logarithmic growth, spermidine N⁸-acetyltransferase activity remained at plateau for 1 day declining thereafter, while spermidine/spermine N¹-acetyltransferase activity immediately decreased. In Yoshida AH-130 hepatoma cells transplanted in rat peritoneum, spermidine N⁸-acetyltransferase and spermidine/spermine N¹-acetyltransferase activities rose, respectively, in concomitance with elevated proliferation-rate and quasi-stationary phase of growth. Since the expression of cytosolic and nuclear acetyltransferases underwent different temporal activation, an involvement of these enzymes in separate metabolic processes controlling normal and neoplastic growth may be suggested.     

Effect of polyamines and cations on the in vitro methylation of histones

Na⁺ (0.05–0.15 M) increases both the rate and extent of methylation of chromosomal bound histone H4, while spermidine markedly inhibits this reaction. The effects of spermidine could be mimicked by increasing the concentration of Mg²⁺ or Ca²⁺ to 5–10 mM. At the concentrations listed above, these cations have no significant effect on the methylation of free or chromosomal bound histone H3, nor do they affect the rate or extent of methylation of soluble histone H4. Apparently, the accessibility of histone H4 to the methyltransferase is influenced by chromatin structure. Increasing concentrations of Na⁺ alter the conformation of chromatin (DNA) in such a way as to expose lysine residues in the N-terminal region of histone H4 to the methyltransferase, whereas Mg²⁺ or spermidine acts in an opposite manner.     

DNA methylation is not involved in the structural alterations of Ornithine Decarboxylase or Total Chromatin of c-Ha-rasVal 12 Oncogene-Transformed NIH-3T3 Fibroblasts

The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c-Ha-ras^{Val 12} oncogene-transformed NIH-3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid-coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene-transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c-Ha-ras^{Val 12} oncogene.     

Interaction between polyamines and nucleic acids or phospholipids

The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K⁺ or Mg²⁺ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 mm Mg²⁺ and 100 mm K⁺, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine.     

Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention

Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N ¹-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N ¹-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

Localization of some molecules within the grooves of DNA by modification of their complexes with dimethyl sulphate

The comparative methylation with [³H] dimethyl sulphate (DMS) of free DNA and DNA in chromatin and nuclei within the minor and major grooves of the DNA double helix and its single stranded regions was measured.The results suggest that histones lie partly inside the major groove and partly out of the grooves of DNA in chromatin leaving the minor groove open; most of the non-histone proteins of chromatin are not buried in the DNA grooves; the content of single stranded DNA in chromatin does not exceed 0.5%.     

Brain infarction correlates more closely with acrolein than with reactive oxygen species

Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from spermine by spermine oxidase. Production of acrolein from spermine by spermine oxidase was clarified using spermine synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which spermine content is negligible or spermidine/spermine N¹-acetyltransferase activity is elevated.