CpG Steps Research Articles

Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson–Crick base pair at the two drug-binding interfaces can result in a single-stranded ‘chair-shaped’ DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a ‘curved-shaped’ duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs’ potential synergetic use.

Lone Pair…π Contacts and Structure Signatures of r(UNCG) Tetraloops, Z-Turns, and Z-Steps: A WebFR3D Survey.

Z-DNA and Z-RNA have long appeared as oddities to nucleic acid scientists. However, their Z-step constituents are recurrently observed in all types of nucleic acid systems including ribosomes. Z-steps are NpN steps that are isostructural to Z-DNA CpG steps. Among their structural features, Z-steps are characterized by the presence of a lone pair…π contact that involves the stacking of the ribose O4′ atom of the first nucleotide with the 3′-face of the second nucleotide. Recently, it has been documented that the CpG step of the ubiquitous r(UNCG) tetraloops is a Z-step. Accordingly, such r(UNCG) conformations were called Z-turns. It has also been recognized that an r(GAAA) tetraloop in appropriate conditions can shapeshift to an unusual Z-turn conformation embedding an ApA Z-step. In this report, we explore the multiplicity of RNA motifs based on Z-steps by using the WebFR3D tool to which we added functionalities to be able to retrieve motifs containing lone pair…π contacts. Many examples that underscore the diversity and universality of these motifs are provided as well as tutorial guidance on using WebFR3D. In addition, this study provides an extensive survey of crystallographic, cryo-EM, NMR, and molecular dynamics studies on r(UNCG) tetraloops with a critical view on how to conduct database searches and exploit their results.

Novel eGZ-motif formed by regularly extruded guanine bases in a left-handed Z-DNA helix as a major motif behind CGG trinucleotide repeats.

The expansion of d(CGG) trinucleotide repeats (TRs) lies behind several important neurodegenerative diseases. Atypical DNA secondary structures have been shown to trigger TR expansion: their characterization is important for a molecular understanding of TR disease. CD spectroscopy experiments in the last decade have unequivocally demonstrated that CGG runs adopt a left-handed Z-DNA conformation, whose features remain uncertain because it entails accommodating GG mismatches. In order to find this missing motif, we have carried out molecular dynamics (MD) simulations to explore all the possible Z-DNA helices that potentially form after the transition from B- to Z-DNA. Such helices combine either CpG or GpC Watson-Crick steps in Z-DNA form with GG-mismatch conformations set as either intrahelical or extrahelical; and participating in BZ or ZZ junctions or in alternately extruded conformations. Characterization of the stability and structural features (especially overall left-handedness, higher-temperature and steered MD simulations) identified two novel Z-DNA helices: the most stable one displays alternately extruded Gs, and is followed by a helix with symmetrically extruded ZZ junctions. The G-extrusion favors a seamless stacking of the Watson-Crick base pairs; extruded Gs favor syn conformations and display hydrogen-bonding and stacking interactions. Such conformations could have the potential to hijack the MMR complex, thus triggering further expansion.

Ethidium bromide interactions with DNA: an exploration of a classic DNA-ligand complex with unbiased molecular dynamics simulations.

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Short but Weak: The Z‐DNA Lone‐Pair⋅⋅⋅π Conundrum Challenges Standard Carbon Van der Waals Radii

AbstractCurrent interest in lone‐pair⋅⋅⋅π (lp⋅⋅⋅π) interactions is gaining momentum in biochemistry and (supramolecular) chemistry. However, the physicochemical origin of the exceptionally short (ca. 2.8 Å) oxygen‐to‐nucleobase plane distances observed in prototypical Z‐DNA CpG steps remains unclear. High‐level quantum mechanical calculations, including SAPT2+3 interaction energy decompositions, demonstrate that lp⋅⋅⋅π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatic interactions combined with stereochemical effects imposed by the locally strained structural context. They also suggest that the carbon van der Waals (vdW) radii, originally derived for sp3 carbons, should not be used for smaller sp2 carbons attached to electron‐withdrawing groups. Using a more adapted carbon vdW radius results in these lp⋅⋅⋅π contacts being no longer of the sub‐vdW type. These findings challenge the whole lp⋅⋅⋅π concept that refers to elusive orbital interactions that fail to explain short interatomic contact distances.

Short but Weak: The Z-DNA Lone-Pair⋅⋅⋅π Conundrum Challenges Standard Carbon Van der Waals Radii.

Current interest in lone-pair⋅⋅⋅π (lp⋅⋅⋅π) interactions is gaining momentum in biochemistry and (supramolecular) chemistry. However, the physicochemical origin of the exceptionally short (ca. 2.8 Å) oxygen-to-nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. High-level quantum mechanical calculations, including SAPT2+3 interaction energy decompositions, demonstrate that lp⋅⋅⋅π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatic interactions combined with stereochemical effects imposed by the locally strained structural context. They also suggest that the carbon van der Waals (vdW) radii, originally derived for sp3 carbons, should not be used for smaller sp2 carbons attached to electron-withdrawing groups. Using a more adapted carbon vdW radius results in these lp⋅⋅⋅π contacts being no longer of the sub-vdW type. These findings challenge the whole lp⋅⋅⋅π concept that refers to elusive orbital interactions that fail to explain short interatomic contact distances.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Structure and Dynamics of DNA and RNA Double Helices Obtained from the CCG and GGC Trinucleotide Repeats.

Expansions of both GGC and CCG sequences lead to a number of expandable, trinucleotide repeat (TR) neurodegenerative diseases. Understanding of these diseases involves, among other things, the structural characterization of the atypical DNA and RNA secondary structures. We have performed molecular dynamics simulations of (GCC) n and (GGC) n homoduplexes in order to characterize their conformations, stability, and dynamics. Each TR has two reading frames, which results in eight nonequivalent RNA/DNA homoduplexes, characterized by CpG or GpC steps between the Watson-Crick base pairs. Free energy maps for the eight homoduplexes indicate that the C-mismatches prefer anti-anti conformations, while G-mismatches prefer anti-syn conformations. Comparison between three modifications of the DNA AMBER force field shows good agreement for the mismatch free energy maps. The mismatches in DNA-GCC (but not CCG) are extrahelical, forming an extended e-motif. The mismatched duplexes exhibit characteristic sequence-dependent step twist, with strong variations in the G-rich sequences and the e-motif. The distribution of Na+ is highly localized around the mismatches, especially G-mismatches. In the e-motif, there is strong Na+ binding by two G(N7) atoms belonging to the pseudo GpC step created when cytosines are extruded and by extrahelical cytosines. Finally, we used a novel technique based on fast melting by means of an infrared laser pulse to classify the relative stability of the different DNA-CCG and -GGC homoduplexes.

Does Cytosine Methylation Stabilize the BI Substate of DNA?

The local conformation of DNA plays a critical role in the recognition-and-binding process of transcription factors. In particular, the most common form of the DNA double helix, B-DNA, exists as a conformational equilibrium between BI and BII substates that are associated with changes in major and minor groove dimensions. It has been hypothesized that cytosine methylation, the most common epigenetic modification and one that is often found in so-called CpG islands, modulates transcription factor binding affinity by altering the BI-BII equilibrium in the neighborhood of methylation sites. Indeed, previous molecular dynamics studies have suggested that cytosine methylation leads to a uniform stabilization of the BI substate in CpG islands. In this work we first benchmark the ability of the latest Amber DNA force fields and a variety of three-point water models to reproduce BI-BII equilibrium in the Dickerson dodecamer and reduce terminal base pair fraying. Using the best-performing combinations, we then re-examine the conformational effects of cytosine methylation in a prototypical CpG island, (GC)5. We find that the BI substate is stabilized for GpC steps in such sequences, but that the BII substate is stabilized for the CpG steps. More interestingly, the Amber ff99bsc0 DNA force field used in previous studies yields opposite conclusions. These findings highlight the need for continued fixed-charge force field development for nucleic acids, as well as solution state experimental datasets involving epigenetic modification that can serve as benchmarks for simulation accuracy.

Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.

Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.

Thioester Bonds of Thiocoraline Can Be Replaced with NMe-Amide Bridges without Affecting Its DNA-Binding Properties.

In the search for new drug candidates for DNA recognition, affinity and sequence selectivity are two of the most important features. NMe-azathiocoraline, a synthetic antitumor bisintercalator derived from the natural marine product thiocoraline, shows similar potency to the parent compound, as well as possessing enhanced stability. Analysis of the DNA-binding selectivity of NMe-azathiocoraline by DNase I footprinting using universal substrates with all 136 tetranucleotides and all possible symmetrical hexanucleotide sequences revealed that, although this ligand binds to all CpG steps with lower affinities than thiocoraline, it displays additional binding to AT-rich sites. Moreover, fluorescence melting studies showed a strong interaction of the synthetic molecule with CACGTG and weaker binding to ACATGT and AGATCT. These findings demonstrate that NMe-azathiocoraline has the same mode of action as thiocoraline, mimicking its DNA-binding selectivity despite the substitution of its thioester bonds by NMe-amide bridges.

Structural fluctuation observed in Z-DNA d(CGCGCG)₂ in the absence of divalent metal cations and polyamines.

In the present study, Z-DNA d(CGCGCG)2 was crystallized from a DNA solution in the absence of divalent metal cations and polyamines, and its X-ray structure was determined at 0.98 Å resolution. Comparison of this structure and previously reported Z-DNA structures, containing Mg(2+) cations and/or polyamines, demonstrated that Z-DNA can have structural fluctuations with respect to phosphate groups and hydration in the minor groove. At the GpC steps, a two-state structural equilibrium between the ZI and ZII conformations was frequently observed. In contrast, at the CpG steps, the phosphate groups exhibited rotational fluctuation, which could induce distortion of sugar puckering. In addition, alternative positions of water molecules were found in the middle of the minor groove of the Z-DNA. These structural fluctuations were likely observable because of the absence of Mg(2+) cations and polyamines. The results related to these phenomena were supported by those of other experimental methods, suggesting the possibility of these fluctuations occurring in biological conditions.

CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI

Cytosine methylation promotes deamination. In eukaryotes, CpG methylation is thought to account for CpG underrepresentation. Whether scarcity of CpGs in prokaryotic genomes is diagnostic for methylation is not clear. Here, we report that Mycoplasms tend to be CpG depleted and to harbor a family of constitutively expressed or phase variable CpG-specific DNA methyltransferases. The very CpG poor Mycoplasma penetrans and its constitutively active CpG-specific methyltransferase M.MpeI were chosen for further characterization. Genome-wide sequencing of bisulfite-converted DNA indicated that M.MpeI methylated CpG target sites both in vivo and in vitro in a locus-nonselective manner. A crystal structure of M.MpeI with DNA at 2.15-Å resolution showed that the substrate base was flipped and that its place in the DNA stack was taken by a glutamine residue. A phenylalanine residue was intercalated into the "weak" CpG step of the nonsubstrate strand, indicating mechanistic similarities in the recognition of the short CpG target sequence by prokaryotic and eukaryotic DNA methyltransferases.

Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation.

DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation.

Rationale for the opposite stereochemistry of the major monoadducts and interstrand crosslinks formed by mitomycin C and its decarbamoylated analogue at CpG steps in DNA and the effect of cytosine modification on reactivity

Mitomycin C (MMC) is a potent antitumour agent that forms a covalent bond with the 2-amino group of selected guanines in the minor groove of double-stranded DNA following intracellular reduction of its quinone ring and opening of its aziridine moiety. At some 5'-CG-3' (CpG) steps the resulting monofunctional adduct can evolve towards a more deleterious bifunctional lesion, which is known as an interstrand crosslink (ICL). MMC reactivity is enhanced when the cytosine bases are methylated (5 MC) and decreased when they are replaced with 5-F-cytosine (5FC) whereas the stereochemical preference of alkylation changes upon decarbamoylation. We have studied three duplex oligonucleotides of general formula d(CGATAAXGCTAACG) in which X stands for C, 5MC or 5FC. Using a combination of molecular dynamics simulations in aqueous solution, quantum mechanics and continuum electrostatics, we have been able to (i) obtain a large series of snapshots that facilitate an understanding in atomic detail of the distinct stereochemistry of monoadduct and ICL formation by MMC and its decarbamoylated analogue, (ii) provide an explanation for the altered reactivity of MMC towards DNA molecules containing 5MC or 5FC, and (iii) show the distinct accommodation in the DNA minor groove of the different covalent modifications, particularly the most cytotoxic C1α and C1β ICLs.

Solvent effects on the conformation of DNA dodecamer segment: A simulation study

Different solvent temperatures with five kinds of counterions are used to investigate solvent effects on the DNA microscopic structure. The dodecamer d (CGCGAATTCGCG) DNA segment is merged into the solvents and its conformation transition is studied with the molecular dynamics simulations in detail. For the simple point charge model of water molecule with Na(+) counterions, as temperature increases from 200 K to 343 K, the duplex DNA changes from stiff B form to a state between A form and B form, which we define as mixed (A-B) structure, with a double helix unwinding. To study the counterions effects, other four alkali cations, Li(+), K(+), Rb(+), or Cs(+) ions, are substituted for Na(+) ions at 298 K and 343 K, respectively. For the cases of Li(+), Rb(+), and Cs(+) ions, the duplex DNA becomes more flexible with sugar configuration changing form C2'-endo to C1'-endo type and the width and depth of minor groove at CpG and GpC steps moving towards A values, as the mass of the counterions decreasing. For the case of K(+) ions, DNA-K(+) interaction widens the width of minor and major grooves at ApA steps and TpT steps, respectively. It seems that the light ions (Li(+) or Na(+)) prefer to interact with the free phosphate oxygen atoms while the heavier ions (Rb(+) and Cs(+)) strongly interact with the base pairs.

Structure of a DNA Repair Substrate Containing an Alkyl Interstrand Cross-Link at 1.65 Å Resolution,

Chemotherapeutic alkylating agents, such as bifunctional nitrogen mustards and cisplatins, generate interstrand DNA cross-links that inhibit cell proliferation by arresting DNA transcription and replication. A synthetic N4C-ethyl-N4C interstrand cross-link between opposing cytidines mimics the DNA damage produced by this class of clinically important compounds and can be synthesized in large quantities to study the repair, physical properties, and structures of these DNA adducts. The X-ray structure of a DNA duplex d(CCAAC*GTTGG)2 containing a synthetic N4C-ethyl-N4C interstrand cross-link between the cytosines of the central CpG step (*) has been determined at 1.65 A resolution. This structure reveals that the ethyl cross-link in the CpG major groove does not significantly disrupt the B-form DNA helix. Comparison of the N4C-ethyl-N4C cross-linked structure with the structure of an un-cross-linked oligonucleotide of the same sequence reveals that the cross-link selectively stabilizes a preexisting alternative conformation. The conformation preferred by the cross-linked DNA is constrained by the geometry of the ethyl group bridging the cytosine amines. Characteristics of the cross-linked CpG step include subtle differences in the roll of the base pairs, optimized Watson-Crick hydrogen bonds, and loss of a divalent cation binding site. Given that the N4C-ethyl-N4C cross-link stabilizes a preexisting conformation of the CpG step, this synthetically accessible substrate presents an ideal model system for studying the genomic effects of covalently coupling the DNA strands, independent of gross alterations in DNA structure.

M.TaqI facilitates the base flipping via an unusual DNA backbone conformation

MD simulations have been carried out to understand the dynamical behavior of the DNA substrate of the Thermus aquaticus DNA methyltransferase (M.TaqI) in the methylation process at N6 of adenine. As starting structures, an x-ray structure of M.TaqI in complex with DNA and cofactor analogue (PDB code: 1G 38) and free decamer d(GTTCGATGTC)(2) were taken. The x-ray structure shows two consecutive BII substates that are not observed in the free decamer. These consecutive BII substates are also observed during our simulation. Additionally, their facing backbones adopt the same conformations. These double facing BII substates are stable during the last 9 ns of the trajectories and result in a stretched DNA structure. On the other hand, protein-DNA contacts on 5' and 3' phosphodiester groups of the partner thymine of flipped adenine have changed. The sugar and phosphate parts of thymine have moved further into the empty space left by the flipping base without the influence of protein. Furthermore, readily high populated BII substates at the GpA step of palindromic tetrad TCGA rather than CpG step are observed in the free decamer. On the contrary, the BI substate at the GpA step is observed on the flipped adenine strand. A restrained MD simulation, reproducing the BI/BII pattern in the complex, demonstrated the influence of the unusual backbone conformation on the dynamical behavior of the target base. This finding along with the increased nearby interstrand phosphate distance is supportive to the N6-methylation mechanism.

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be −3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.

Structure, Flexibility, and Repair of Two Different Orientations of the Same Alkyl Interstrand DNA Cross-Link

Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.