Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)₄ motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.     

Discrimination of single-nucleotide alterations by G-specific fluorescence quenching

A new strategy for the detection of single-base alterations through fluorescence quenching by guanine (G) is described. We have devised a novel base-discriminating fluorescent (BDF) nucleoside, 4'PyT, that contains a pyrenecarboxamide fluorophore at the thymidine sugar's C4'-position. 4'PyT-containing oligodeoxynucleotides only exhibited enhanced fluorescence in response to the presence of a complementary adenine base. In contrast, the fluorescence of mismatched duplexes containing 4'PyT/N base pairs (N = C, G, or T) was considerably weaker. This highly A-selective fluorescence was a product of guanine-specific quenching efficiency; when the complementary base to 4'PyT was a mismatch, the pyrenecarboxamide fluorophore was able to interact intimately with neighboring G bases (the most likely interaction in the case of intercalation), so effective quenching by the G bases occurred in the mismatched duplexes. In contrast, duplexes containing 4'PyT/A base pairs exhibited strong emission, since in this case the fluorophores were positioned in the minor groove and able to escape fluorescence quenching by the G bases.     

High‐Fidelity Recognition of RNA: Solution Structure of a DNA:RNA Hybrid Duplex with a Molecular Cap

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson–Crick pairing at their termini. Here we report the three‐dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5′ terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard‐to‐suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high‐fidelity probes for RNA.     

Development of Polyamine-Substituted Triphenylamine Ligands with High Affinity and Selectivity for G-Quadruplex DNA

Currently, significant efforts are devoted to designing small molecules able to bind selectively to guanine quadruplexes (G4s). These noncanonical DNA structures are implicated in various important biological processes and have been identified as potential targets for drug development. Previously, a series of triphenylamine (TPA)-based compounds, including macrocyclic polyamines, that displayed high affinity towards G4 DNA were reported. Following this initial work, herein a series of second-generation compounds, in which the central TPA has been functionalised with flexible and adaptive linear polyamines, are presented with the aim of maximising the selectivity towards G4 DNA. The acid–base properties of the new derivatives have been studied by means of potentiometric titrations, UV/Vis and fluorescence emission spectroscopy. The interaction with G4s and duplex DNA has been explored by using FRET melting assays, fluorescence spectroscopy and circular dichroism. Compared with previous TPA derivatives with macrocyclic substituents, the new ligands reported herein retain the G4 affinity, but display two orders of magnitude higher selectivity for G4 versus duplex DNA; this is most likely due to the ability of the linear substituents to embrace the G4 structure.     

Structure-function relationships of phenoxazine nucleosides for identification of mismatches in duplex DNA by fluorescence spectroscopy

The effects of the flanking sequence on the mismatch‐detection capabilities of the fluorescent nucleoside phenoxazine ( tC^O ) were examined in a systematic fashion, and compared to the previously reported fluorescent, phenoxazine‐based nucleoside Ç^f . We see some similarities for the two fluorescent nucleosides, for example, the emission intensity of the C‐mismatched duplex is always the highest, and a three‐peak pattern in the spectrum emerges when the fluorosides are base‐paired with A. However, phenoxazine was only able to distinguish a mismatch from the fully base‐paired duplex in 11 out of 16 flanking sequences, and was able to identify each of the mismatches in six of those sequences. Therefore, tC^O shows poorer discrimination of mismatches than was previously reported for Ç^f , which could be used to identify all base‐pairing partners in all immediately flanking sequences, albeit in some cases by using mercuric ions to selectively quench the emission of the T‐mismatched duplex. The mercuric titration might resolve the overlap of fluorescence curves of tC^O in some flanking sequences, but not for 5′‐d(C tC^O G) and 5′‐d(T tC^O A) due to overlap of A‐mismatch and G‐match fluorescence curves. A pH titration was performed on Ç^f , tC^O and a N5‐methylated derivative of tC^O , which showed that the emergence of the three‐peak pattern is associated with the de‐protonation of N5 in the fluorosides. We also show that neither the α‐ nor β‐anomer of the phenothiazine nucleoside ( tC ) was able to detect a mismatch in any of the flanking sequences examined.     

DNA mismatch-specific base flipping by a bisacridine macrocycle

Most, if not all, enzymes that chemically modify nucleobases in DNA flip their target base from the inside of the double helix into an extrahelical position. This energetically unfavorable conformation is partly stabilized by specific binding of the apparent abasic site being formed. Thus, DNA base-flipping enzymes, like DNA methyltransferases and DNA glycosylases, generally bind very strongly to DNA containing abasic sites or abasic-site analogues. The macrocyclic bisacridine BisA has previously been shown to bind abasic sites. Herein we demonstrate that it is able to specifically recognize DNA base mismatches and most likely induces base flipping. Specific binding of BisA to DNA mismatches was studied by thermal denaturation experiments by using short duplex oligodeoxynucleotides containing central TT, TC, or TG mismatches or a TA match. In the presence of the macrocycle a strong increase in the melting temperature of up to 7.1 degrees C was observed for the mismatch-containing duplexes, whereas the melting temperature of the fully matched duplex was unaffected. Furthermore, BisA binding induced an enhanced reactivity of the mispaired thymine residue in the DNA toward potassium permanganate oxidation. A comparable reactivity has previously been observed for a TT target base mismatch in the presence of DNA methyltransferase M.TaqI. This similarity to a known base-flipping enzyme suggests that insertion of BisA into the DNA helix displaces the mispaired thymine residue into an extrahelical position, where it should be more prone to chemical oxidation. Thus, DNA base flipping does not appear to be limited to DNA-modifying enzymes but it is likely to also be induced by a small synthetic molecule binding to a thermodynamically weakened site in DNA.     

Structure and Replication of yDNA: A Novel Genetic Set Widened by Benzo‐Homologation

In a functioning genetic system, the information‐encoding molecule must form a regular self‐complementary complex (for example, the base‐paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo‐widened DNA‐like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D‐NMR methods for a simple sequence composed of T–yA/yA–T pairs. The data confirm an antiparallel, right‐handed, hydrogen‐bonded helix resembling B‐DNA but with a wider diameter and enlarged base‐pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T–yA, A–yT, and G–yC are measured by steady‐state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base‐pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T–yT and T–yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information‐encoding molecule and as a tool for application in basic science and biomedicine.     

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum,a member of the endonuclease III family of repair proteins,excises the pyrimidine base from T–G and U–G mismatches.Unlike endonuclease III, it does not cleave the phosphodiesterbackbone by a ß-elimination reaction. This cleavage eventhas been attributed to a nucleophilic attack by the conservedLys120 of endonuclease III on the aldehyde group at C1' of thedeoxyribose and subsequent Schiff base formation. The inabilityof TDG to perform this ß-elimination event appears tobe due to the presence of a tyrosine residue at the positionequivalent to Lys120 in endonuclease III. The purpose of thiswork was to investigate the requirements for AP lyase activity.We replaced Tyr126 in TDG with a lysine residue to determineif this replacement would yield an enzyme with an associatedAP lyase activity capable of removing a mismatched pyrimidine.We observed that this replacement abolishes the glycosylaseactivity of TDG but does not affect substrate recognition. Itdoes, however, convert the enzyme into an AP lyase. Chemicaltrapping assays show that this cleavage proceeds through a Schiffbase intermediate and suggest that the amino acid at position126 interacts with C1' on the deoxyribose sugar.     

氨基酸类希夫碱配合物的性能及对DNA作用机理研究

氨基酸希夫碱配合物是一类生物活性物质,具有抗肿瘤、杀菌、抑制癌细胞作用,其衍生物可作为人造核酸酶和有效的DNA切割试剂。这类配合物与DNA的相互作用可以从分子水平认识特定DNA系列、改变DNA结构和影响基因表达过程进行医学治疗诊断。介绍了醛缩、酮缩氨基酸希夫碱配合物的类型、合成方法、性质和生物作用。分析了氨基酸希夫碱参与过渡金属、稀土金属配位成键的方式和空间构型。讨论了标题化合物与DNA之间的作用机理。     

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases

Spectroscopic characterization of Ag^I-ion-mediated C-Ag^I-A and C-Ag^I-T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by Ag^I ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of Ag^I ions is in the order C-A≈C-T>C-C, the stabilizing effect of Ag^I ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to Ag^I-mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.     

Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd2+ Chemical Stimulus

The on‐column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio‐directed Cd²⁺ coordination; a route for healing DNA damage. Short oligomers are G‐specifically thiolated through a modified on‐column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd²⁺ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3′‐A₆tG₄T₆‐5′, suggests a (tG)₈Cd₂ central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal‐induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd²⁺ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer.     

Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase

The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.     

Synthesis and in Vitro Evaluation of Novel 5-Nitroindole Derivatives as c-Myc G-Quadruplex Binders with Anticancer Activity

Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.     

Directed DNA polymerase evolution: effects of mutations in motif C on the mismatch-extension selectivity of thermus aquaticus DNA polymerase

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.     

Novel DNA Catalysts Based on G‐Quadruplex for Organic Synthesis

Double‐stranded DNA (dsDNA) as a catalytic species has been applied in various key asymmetric reactions. Compared with dsDNA, G‐quadruplex DNAs (G4DNAs) have unique conformational structures and versatile complexation properties, and act expectedly as hosts to recognize planar aromatic molecules through π–π stacking. Based on the specific recognition for small molecules, G4DNA‐based catalysts have been used to assist the catalytic function, and have been applied in asymmetric organic reactions.

     

Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases MUTYH and hOGG1 in Colorectal Cancer Patients

Oxidative stress, oxidative DNA damage and resulting mutations play a role in colorectal carcinogenesis. Impaired equilibrium between DNA damage formation, antioxidant status, and DNA repair capacity is responsible for the accumulation of genetic mutations and genomic instability. The lesion-specific DNA glycosylases, e.g., hOGG1 and MUTYH, initiate the repair of oxidative DNA damage. Hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome) with germline mutations causing a loss-of-function in base excision repair glycosylases, serve as straight forward evidence on the role of oxidative DNA damage and its repair. Altered or inhibited function of above glycosylases result in an accumulation of oxidative DNA damage and contribute to the adenoma-adenocarcinoma transition. Oxidative DNA damage, unless repaired, often gives rise G:C > T:A mutations in tumor suppressor genes and proto-oncogenes with subsequent occurrence of chromosomal copy-neutral loss of heterozygosity. For instance, G>T transversions in position c.34 of a KRAS gene serves as a pre-screening tool for MUTYH-associated polyposis diagnosis. Since sporadic colorectal cancer represents more complex and heterogenous disease, the situation is more complicated. In the present study we focused on the roles of base excision repair glycosylases (hOGG1, MUTYH) in colorectal cancer patients by investigating tumor and adjacent mucosa tissues. Although we found downregulation of both glycosylases and significantly lower expression of hOGG1 in tumor tissues, accompanied with G>T mutations in KRAS gene, oxidative DNA damage and its repair cannot solely explain the onset of sporadic colorectal cancer. In this respect, other factors (especially microenvironment) per se or in combination with oxidative DNA damage warrant further attention. Base excision repair characteristics determined in colorectal cancer tissues and their association with disease prognosis have been discussed as well.