In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases

The expansion of DNA repeat sequences is associated with many genetic diseases in humans. Simple bulge DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. To probe the possible role of bulged structures in DNA slippage, we designed and synthesized a pair of simple chiral spirocyclic compounds [Xi Z, Ouyang D & Mu HT (2006) Bioorg Med Chem Lett 16, 1180-1184], DDI-1A and DDI-1B, which mimic the molecular architecture of the enediyne antitumor antibiotic neocarzinostatin chromophore. Both compounds strongly stimulated slippage in various DNA repeats in vitro. Enhanced slippage synthesis was found to be synchronous for primer and template. CD spectra and UV thermal stability studies supported the idea that DDI-1A and DDI-1B exhibited selective binding to the DNA bulge and induced a significant conformational change in bulge DNA. The proposed mechanism for the observed in vitro expansion of long DNA is discussed.     

Stimulation of DNA strand slippage synthesis by a bulge binding synthetic agent

It has been postulated that bulged structures may be intermediates in the DNA strand slippage synthesis associated with the expansion of nucleotide repeats in various neurodegenerative diseases and cancer. To probe the possible role of bulged structures in this process, we have synthesized a wedge-shaped spirocyclic molecule, DDI (double-decker intercalator), on the basis of our earlier work with the bulge-specific derivative prepared from the enediyne antitumor antibiotic neocarzinostatin chromophore. Using a series of primers/templates containing nucleotide repeats [(AAT)(3)/(ATT)(5), (ATT)(3)/(AAT)(5), (CAG)(3)/(CTG)(5), (CA)(4)C/(GT)(7)G, (GT)(4)G/(CA)(7)C, T(9)/A(30), T(20)/A(30)] with the Klenow fragment of Escherichia coli DNA polymerase I, we find that DDI markedly enhances the formation of long DNA products, whose synthesis would require strand slippage to occur. DDI-induced slippage synthesis is more pronounced as the incubation proceeds and at limiting enzyme levels. The gel band pattern of the synthesized DNA products reflects the particular nucleotide repeat unit and is not altered by DDI. The lack of any drug effect on primer extension on M13 DNA and heteropolymeric 62-mer templates, where strand slippage is much less likely to occur, suggests that stimulation of slippage synthesis by DDI is not due to a direct effect on the enzyme. By contrast, other DNA-binding agents, such as ethidium bromide, distamycin, and doxorubicin, inhibit the formation of slippage-induced DNA products, but this block can be overcome by DDI, presumably by its destabilizing duplex DNA-binding sites for these other agents. We propose that DDI binds to or induces the formation of a bulge or related structure, which promotes DNA strand slippage and its consequent expansion of nucleotide repeats during replication by DNA polymerase I and that this action provides insight into the development of agents that interfere with nucleotide expansions found in various disease states.     

A bulge binding agent with novel wedge-shape topology for stimulation of DNA triplet repeat strand slippage synthesis

Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Herein a bulge binding agent with novel wedge-shape topology of the aromatic moiety was designed and synthesized. The compound-bulge DNA interactions were characterized via UV melting experiments, circular dichroism and were quantitated by surface plasmon resonance with K(d) of 41.5 microM. This compound showed remarkable stimulation for DNA triplet repeat strand slippage synthesis in vitro.     

Interaction of bulged DNA with leucine-containing mimics of NCS-chrom

Synthesis of chiral spirocyclic helical compounds containing leucine that mimic the molecular architecture of the potent DNA bulge binder obtained from the natural product metabolite NCSi-gb has been accomplished. The interaction between the compounds and DNA was studied by circular dichroism (CD) method. The results suggested that the two synthetic diastereoisomers specifically targeted the bulge site of DNA and induced conformational change of bulged DNA greatly.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

Solution structure of a wedge-shaped synthetic molecule at a two-base bulge site in DNA

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes.     

Designed DNA probes from the neocarzinostatin family: Impact of glycosyl linkage stereochemistry on bulge base binding

Bulged sites in DNA and RNA have become targets for rational drug design due to their suspected involvement in a number of key biomolecular processes. A lead compound, derived from the enediyne natural product NCS-chrom has been used to inform chemical synthesis of a family of designed probes of DNA bulges, one of which shows 80 nM affinity for a two base bulged target. Key contributors to binding of these spirocyclic compounds have been studied in order to correlate affinity and specificity with structural features. Herein, we demonstrate that the glycosyl linkage stereochemistry of the pendant aminofucosyl group plays a pivotal role in binding, and coupled with insight obtained with various bulged targets, will allow rational design of second generation ligands.     

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.     

Different mechanisms inferred from sequences of human mitochondrial DNA deletions in ocular myopathies.

We have sequenced the deletion borders of the muscle mitochondrial DNA from 24 patients with heteroplasmic deletions. The length of these deletions varies from 2.310 bp to 8.476 bp and spans from position 5.786 to 15.925 of the human mitochondrial genome preserving the heavy chain and light chain origins of replication. 12 cases are common deletions identical to the mutation already described by other workers and characterized by 13 bp repeats at the deletion boundaries, one of these repeats being retained during the deletion process. The other cases (10 out of 12) have shown deletions which have not been previously described. All these deletions are located in the H strand DNA region which is potentially single stranded during mitochondrial DNA replication. In two cases, the retained Adenosine from repeat closed to the heavy strand origin of replication would indicate slippage mispairing. Furthermore in one patient two mt DNA molecules have been cloned and their sequences showed the difference of four nucleotides in the breakpoint of the deletion, possibly dued to slippage mispairing. Taken together our results suggest that deletions occur either by slippage mispairing or by internal recombination at the direct repeat level. They also suggest that different mechanisms account for the deletions since similarly located deletions may display different motives at the boundaries including the absence of any direct repeat.     

In vitro replication slippage by DNA polymerases from thermophilic organisms

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.     

Stereochemical control of small molecule binding to bulged DNA: comparison of structures of spirocyclic enantiomer-bulged DNA complexes

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and ent-DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. Ent-DDI is a left-handed wedge-shaped spirocyclic molecule whose aglycone portion is an enantiomer of DDI, which mimics the spirocyclic geometry of the natural product, NCSi-gb, formed by base-catalyzed activation of the enediyne antibiotic neocarzinostatin. The benzindanone moiety of ent-DDI intercalates between the A6.T21 and the T9.A20 base pairs, overlapping with portions of the purine bases; the dihydronaphthalenone moiety is positioned in the minor groove along the G7-T8-T9 bulge sequence; and the aminoglycoside is in the middle of the minor groove, approaching A20 of the nonbulged strand. This alignment of ent-DDI along the DNA helical duplex is in the reverse direction to that of DDI. The aminoglycoside moiety of ent-DDI is positioned in the 3' direction from the bulge region, whereas that of the DDI is positioned in the 5' direction from the same site. This reverse binding orientation within the bulge site is the natural consequence of the opposite handedness imposed by the spirocyclic ring junction and permits the aromatic ring systems of the two spirocyclic enantiomers access to the bulge region. NMR and CD data indicate that the DNA in the DDI-bulged DNA complex undergoes a larger conformational change upon complex formation in comparison to the ent-DDI-bulged DNA, explaining the different binding affinities of the two drugs to the bulged DNA. In addition, there are different placements of the bulge bases in the helical duplex in the two complexes. One bulge base (G7) stacks inside the helix, and the other one (T8) is extrahelical in the DDI-bulged DNA complex, whereas both bulge bases in the ent-DDI-bulged DNA complex prefer extrahelical positions for drug binding. Elucidation of the detailed binding characteristics of the synthetic spirocyclic enantiomers provides a rational basis for the design of stereochemically controlled drugs for bulge binding sites.     

Molecular probes of DNA bulges: functional assay and spectroscopic analysis

Bulged structures in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these nucleic acid targets would be of considerable utility to chemical biologists. Herein, we report guided chemical synthesis of small molecules capable of binding to DNA bulges by virtue of their unique (spirocyclic) geometry. The agents, modeled on a natural product congener, show pronounced selectivity for specific bulged motifs and are able to enhance slipped DNA synthesis, a hallmark functional assay of bulge binding. Significantly, bulge-agent complexes demonstrate characteristic fluorescent signatures depending on bulge and flanking sequence in the oligo. It is anticipated that these signature patterns can be harnessed as molecular probes of bulged hotspots in DNA and RNA.     

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 1: identification of 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane as lead scaffolds

Herein we report the identification of two new fatty acid amide hydrolase (FAAH) inhibitor lead series with FAAH k(inact)/K(i) potency values greater than 1500M(-1)s(-1). The two novel spirocyclic cores, 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, clearly distinguished themselves from the other spirocyclic cores on the basis of their superior potency for FAAH. Lead compounds from these two series have suitable FAAH potency and selectivity for additional medicinal chemistry optimization.     

Replication strand preference for deletions associated with DNA palindromes

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E . coli , in bacteriophage λ and in high-copy-number pUC-based plasmids.     

Probing DNA bulges with designed helical spirocyclic molecules

Because bulged structures (unpaired bases) in nucleic acids are of general biological significance, it has been of interest to design small molecules as specific probes of bulge function. On the basis of our earlier work with the specific DNA bulge-binding metabolite obtained from the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom), we have prepared three small helical spirocyclic molecules that most closely mimic the natural product. These wedge-shaped molecules resemble the natural product in having the sugar residue attached to the same five-membered ring system. In one instance, the sugar is aminoglucose in beta-glycosidic linkage, and in the other, two enantiomers have the natural sugar N-methylfucosamine in alpha-glycosidic linkage. All three analogues were found to interfere with bulge-specific cleavage by NCS-chrom and the ability of bulged DNA to serve as a template for DNA polymerase 1 in accordance with their binding affinities for DNA containing a two-base bulge. Comparable results were obtained with the analogues for the less efficiently cleaved three-base bulge DNA structures. In each situation, the enantiomers possessing the natural sugar in alpha-glycosidic linkage are the most potent inhibitors of the cleavage reaction. In the DNA polymerase reactions, again, the closest natural product mimics were the most effective in selectively impeding nucleotide extension at the bulge site, presumably by complex formation. These results demonstrate the potential usefulness of bulge-binding compounds in modifying DNA structure and function and support efforts to design and prepare reactive species of these molecules that can covalently modify bulged DNA.     

Replication slippage involves DNA polymerase pausing and dissociation

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.     

Polyethylelimine Masks Mitogenic Effect of DNA

Abstract

The effect of polyethylenimine on the mitogenic stimulation of murine splenocytes by DNA and oligonucleotides has been investigated. It was found that polyethylenimine masks the mitogenic stimulation effect of the compounds.     

Yeast-based assay for the measurement of positive and negative influences on microsatellite stability

The evolutionary conservation of mismatch repair and Saccharomyces cerevisiae as a model system have been exploited for monitoring the influence of everyday beverages and the antineoplastic agent, hydroxyurea, on the stability of regions of highly repetitive DNA known as microsatellites. Two different reporter systems are compared for sensitivity and reproducibility by measuring the extent of frame slippage events occurring in microsatellite regions in wild-type and mismatch repair-compromised yeast strains. Increased frame slippage results in increased reporter gene expression and hence represents instability within the repetitive region, whereas a decrease or no significant change indicates the faithful replication of the original assay plasmid, suggesting a beneficial or neutral effect of the test component. A significant outcome of this study was the identification of the protective influence exerted by the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) against microsatellite instability, which is in agreement with the hypothesis that EGCG is the major chemopreventive ingredient of green tea. Immunological detection can also be used in conjunction with the green fluorescent protein (GFP) version of the assay system to identify compounds, such as hydroxyurea, which increased microsatellite instability. The system has the potential for development as a high-throughput assay for wider application.     

Novel FFA1 (GPR40) agonists containing spirocyclic periphery: polar azine periphery as a driver of potency

A series of nine compounds based on 3-[4-(benzyloxy)phenyl]propanoic acid core containing a 1-oxa-9-azaspiro[5.5]undecane periphery was designed, synthesized and evaluated as free fatty acid 1 (FFA1 or GPR40) agonists. The spirocyclic appendages included in these compounds were inspired by LY2881835, Eli Lilly’s advanced drug candidate for type II diabetes mellitus that was in phase I clinical trials. These polar spirocyclic, fully saturated appendages (that are themselves uncharacteristic of the known FFA1 ligand space) were further decorated with diverse polar groups (such as basic heterocycles or secondary amides). To our surprise, while seven of nine compounds were found to be inactive (likely due to the decrease in lipophilicity, which is known to be detrimental to FFA1 ligand affinity), two compounds containing 2-pyridyloxy and 2-pyrimidinyloxy groups were found to have EC₅₀ of 1.621 and 0.904?µM, respectively. This result is significant in the context of the worldwide quest for more polar FFA1 agonists, which would be devoid of liver toxicity effects earlier observed for a FFA1 agonist fasiglifam (TAk-875) in clinical studies.