The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5′-GA dinucleotides and sequences that contained 5′-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.     

A chimeric satellite transgene sequence is inefficiently targeted by viroid-induced DNA methylation in tobacco

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans.     

Binding of 9-aminoacridine to deoxydinucleoside phosphates of defined sequence: Preferences and stereochemistry

The effect of sequence on the binding of 9-aminoacridine to DNA has been investigated by studying its interaction with deoxydinucleoside phosphates of different sequences using proton nuclear magnetic resonance. Quantitative binding information can be obtained by comparison of the proton chemical shift behavior of 9-aminoacridine upon addition of dinucleoside phosphate to various models for the interaction using least-squares computer fitting procedures. The simplest model that fits the data includes (1) dimerization of 9-aminoacridine and (2) a mixture of 1:1 and 2:1 (dinucleoside phosphate/9-aminoacridine) complexes. The computed parameters allow comparison of binding constants and stereochemistry for different sequences. The 1:1 complexes seem to involve interaction of the ring nitrogen with the backbone phosphate and stacking of one or both chromophores on the acridine; preference in binding is observed for alternating (purine-pyrimidine or pyrimidine-purine) over non-alternating (purine-purine) dinucleoside phosphates. The 2:1 complexes involve intercalation of the acridine between two complementary dinucleoside phosphate strands with weak sequence preferences in binding. The stereochemistry of intercalation differs between non-alternating purine-purine sequences and the alternating pyrimidine-purine or purine-pyrimidine sequences in having the 9-aminoacridine stacked with the purines of one strand rather than straddling the purines on opposite strands. The difference in stereochemistry could possibly be a determining factor in frameshift sequence specificity.     

Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide.

A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes.     

Cytotoxicity and DNA damage induced by a new platinum(II) complex with pyridine and dithiocarbamate

A new platinum(II) complex containing a pyridine nucleus and a dithiocarbamate moiety as ligands ([Pt(ESDT)(Py)Cl]) was evaluated for in vitro cytotoxicity in the cisplatin-sensitive human ovarian 2008 and in the isogenic-resistant C13* cell lines. In both cell types, a tumor cell growth inhibition greater than cisplatin and a complete lack of cross-resistance in C13* cells were found. Despite its molecular size, [Pt(ESDT)(Py)Cl] accumulation was much higher than cisplatin both in parent and resistant cells. Studying the mechanism of action in cell-free media, we established that [Pt(ESDT)(Py)Cl] more efficiently interacts with DNA in vitro compared to cisplatin maintaining a binding preference for GG rich sequences of DNA. On the contrary, DNA platination in vivo by [Pt(ESDT)(Py)Cl] was found lower than cisplatin. An analysis of the type of DNA lesions induced by [Pt(ESDT)(Py)Cl] suggests that the cytotoxic efficacy and the ability to overcome cisplatin resistance seem to be related to a different mechanism of interaction with DNA and/or with other key cellular components.     

Effect of CpG methylation on RAG1/RAG2 reactivity: implications of direct and indirect mechanisms for controlling V(D)J cleavage

It has been suggested that DNA methylation/demethylation is involved in regulating V(D)J rearrangement. Although methylated DNA is thought to induce an inaccessible chromatin structure, it is unclear whether DNA methylation can directly control V(D)J recombination independently of chromatin structure. In this study, we tested whether DNA methylation directly affects the reactivity of the RAG1/RAG2 complex. Specific methylation within the heptamer of the recombination signal sequences (RSS) markedly reduced V(D)J cleavage without inhibiting RAG1/RAG2–DNA complex formation. By contrast, methylation at other positions around the RSS did not affect the reactivity of the RAG proteins. The presence of a methyl-CpG binding-domain protein inhibited the binding of the RAG1/RAG2 complex to all the methylated CpG sites that were tested. Our findings suggest that DNA methylation around the RSS may have a previously unexpected function in regulating V(D)J recombination by directly inhibiting V(D)J cleavage, in addition to its general function of inducing an inaccessible chromatin configuration.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

The SRA methyl-cytosine-binding domain links DNA and histone methylation

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.     

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.     

Methylation pattern of Activator transposase binding sites in maize endosperm.

The maize transposable element Activator (Ac) transposes after replication from only one of the two daughter chromatids. It has been suggested that DNA methylation in conjunction with methylation-sensitive transposase binding to DNA may control the association of Ac transposition and replication. We present here a detailed genomic sequencing analysis of the cytosine methylation patterns of the transposase binding sites within both Ac ends in the wx-m9::Ac allele, where Ac is inserted into the tenth exon of the Waxy gene. The Ac elements in wx-m9::Ac kernels exhibit intriguing methylation patterns and fall into two distinct groups. Approximately 50% of the elements are fully unmethylated at cytosine residues through the 256 nucleotides at the 5' end (the promoter end). The other half is partially methylated between Ac residues 27 and 92. In contrast, at the 3' end, all Ac molecules are heavily methylated between residues 4372 and 4554. The more internally located Ac sequences and the flanking Waxy DNA are unmethylated. Although most methylated cytosines in Ac are in the symmetrical CpG and CpNpG arrangements, nonsymmetrical cytosine methylation is also common in the hypermethylated regions of Ac. These results suggest a model in which differential activation of transposon ends by hemimethylation controls the chromatid selectivity of transposition and the association with replication.     

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.