A Conformational Switch in the Zinc Finger Protein Kaiso Mediates Differential Readout of Specific and Methylated DNA Sequences.

Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to the different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against noncognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.

CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso.

Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression.

Transient Hoogsteen base pairs in canonical duplex DNA.

Sequence-directed variations in the canonical DNA double helix structure that retain Watson-Crick base-pairing have important roles in DNA recognition, topology and nucleosome positioning. By using nuclear magnetic resonance relaxation dispersion spectroscopy in concert with steered molecular dynamics simulations, we have observed transient sequence-specific excursions away from Watson-Crick base-pairing at CA and TA steps inside canonical duplex DNA towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. The observation of Hoogsteen base pairs in DNA duplexes specifically bound to transcription factors and in damaged DNA sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond that which can be achieved based on Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient low-populated nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.

Modulating RNA Alignment Using Directional Dynamic Kinks: Application in Determining an Atomic-Resolution Ensemble for a Hairpin using NMR Residual Dipolar Couplings.

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 µs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps-ns time scales as verified by measurements of R(1ρ) relaxation-dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.

Guanine to inosine substitution leads to large increases in the population of a transient G·C Hoogsteen base pair.

We recently showed that Watson-Crick base pairs in canonical duplex DNA exist in dynamic equilibrium with G(syn)·C+ and A(syn)·T Hoogsteen base pairs that have minute populations of ∼1%. Here, using nuclear magnetic resonance R1ρ relaxation dispersion, we show that substitution of guanine with the naturally occurring base inosine results in an ∼17-fold increase in the population of transient Hoogsteen base pairs, which can be rationalized by the loss of a Watson-Crick hydrogen bond. These results provide further support for transient Hoogsteen base pairs and demonstrate that their population can increase significantly upon damage or chemical modification of the base.

Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy.

Many recently discovered noncoding RNAs do not fold into a single native conformation but sample many different conformations along their free-energy landscape to carry out their biological function. Here we review solution-state NMR techniques that measure the structural, kinetic and thermodynamic characteristics of RNA motions spanning picosecond to second timescales at atomic resolution, allowing unprecedented insights into the RNA dynamic structure landscape. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering.

Characterizing the protonation state of cytosine in transient G·C Hoogsteen base pairs in duplex DNA.

G·C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication, and repair. G·C Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here, we combine NMR and constant pH molecular dynamics simulations to directly investigate the pKa of cytosine N3 in a chemically trapped N1-methyl-G·C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson-Crick geometry at pH >9. A four-state analysis of the pH titration profiles yields a lower bound pKa estimate of 7.2 ± 0.1 for the G·C Hoogsteen base pair, which is in good agreement with the pKa value (7.1 ± 0.1) calculated independently using constant pH MD simulations. Based on these results and pH-dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7-8), G·C Hoogsteen base pairs in naked DNA have a population of 0.02-0.002%, as compared to 0.4% for A·T Hoogsteen base pairs, and likely exist primarily as protonated species.

A historical account of Hoogsteen base-pairs in duplex DNA.

In 1957, a unique pattern of hydrogen bonding between N3 and O4 on uracil and N7 and N6 on adenine was proposed to explain how poly(rU) strands can associate with poly(rA)-poly(rU) duplexes to form triplexes. Two years later, Karst Hoogsteen visualized such a noncanonical A-T base-pair through X-ray analysis of co-crystals containing 9-methyladenine and 1-methylthymine. Subsequent X-ray analyses of guanine and cytosine derivatives yielded the expected Watson-Crick base-pairing, but those of adenine and thymine (or uridine) did not yield Watson-Crick base-pairs, instead favoring "Hoogsteen" base-pairing. More than two decades ensued without experimental "proof" for A-T Watson-Crick base-pairs, while Hoogsteen base-pairs continued to surface in AT-rich sequences, closing base-pairs of apical loops, in structures of DNA bound to antibiotics and proteins, damaged and chemically modified DNA, and in polymerases that replicate DNA via Hoogsteen pairing. Recently, NMR studies have shown that base-pairs in duplex DNA exist as a dynamic equilibrium between Watson-Crick and Hoogsteen forms. There is now little doubt that Hoogsteen base-pairs exist in significant abundance in genomic DNA, where they can expand the structural and functional versatility of duplex DNA beyond that which can be achieved based only on Watson-Crick base-pairing. Here, we provide a historical account of the discovery and characterization of Hoogsteen base-pairs, hoping that this will inform future studies exploring the occurrence and functional importance of these alternative base-pairs.

Structural Plasticity of Pioneer Factor Sox2 and DNA Bendability Modulate Nucleosome Engagement and Sox2-Oct4 Synergism.

Pioneer transcription factors (pTFs) can bind directly to silent chromatin and promote vital transcriptional programs. Here, by integrating high-resolution nuclear magnetic resonance (NMR) spectroscopy with biochemistry, we reveal new structural and mechanistic insights into the interaction of pluripotency pTFs and functional partners Sox2 and Oct4 with nucleosomes. We find that the affinity and conformation of Sox2 for solvent-exposed nucleosome sites depend strongly on their position and DNA sequence. Sox2, which is partially disordered but becomes structured upon DNA binding and bending, forms a super-stable nucleosome complex at superhelical location +5 (SHL+5) with similar affinity and conformation to that with naked DNA. However, at suboptimal internal and end-positioned sites where DNA may be harder to deform, Sox2 favors partially unfolded and more dynamic states that are encoded in its intrinsic flexibility. Importantly, Sox2 structure and DNA bending can be stabilized by synergistic Oct4 binding, but only on adjacent motifs near the nucleosome edge and with the full Oct4 DNA-binding domain. Further mutational studies reveal that strategically impaired Sox2 folding is coupled to reduced DNA bending and inhibits nucleosome binding and Sox2-Oct4 cooperation, while increased nucleosomal DNA flexibility enhances Sox2 association. Together, our findings fit a model where the site-specific DNA bending propensity and structural plasticity of Sox2 govern distinct modes of nucleosome engagement and modulate Sox2-Oct4 synergism. The principles outlined here can potentially guide pTF site selection in the genome and facilitate interaction with other chromatin factors or chromatin opening in vivo.

Probing sequence-specific DNA flexibility in a-tracts and pyrimidine-purine steps by nuclear magnetic resonance (13)C relaxation and molecular dynamics simulations.

Sequence-specific DNA flexibility plays a key role in a variety of cellular interactions that are critical for gene packaging, expression, and regulation, yet few studies have experimentally explored the sequence dependence of DNA dynamics that occur on biologically relevant time scales. Here, we use nuclear magnetic resonance (NMR) carbon spin relaxation combined with molecular dynamics (MD) simulations to examine the picosecond to nanosecond dynamics in a variety of dinucleotide steps as well as in varying length homopolymeric A(n)·T(n) repeats (A(n)-tracts, where n = 2, 4, or 6) that exhibit unusual structural and mechanical properties. We extend the NMR spin relaxation time scale sensitivity deeper into the nanosecond regime by using glycerol and a longer DNA duplex to slow overall tumbling. Our studies reveal a structurally unique A-tract core (for n > 3) that is uniformly rigid, flanked by junction steps that show increasing sugar flexibility with A-tract length. High sugar mobility is observed at pyrimidine residues at the A-tract junctions, which is encoded at the dinucleotide level (CA, TG, and CG steps) and increases with A-tract length. The MD simulations reproduce many of these trends, particularly the overall rigidity of A-tract base and sugar sites, and suggest that the sugar-backbone dynamics could involve transitions in sugar pucker and phosphate backbone BI ↔ BII equilibria. Our results reinforce an emerging view that sequence-specific DNA flexibility can be imprinted in dynamics occurring deep within the nanosecond time regime that is difficult to characterize experimentally at the atomic level. Such large-amplitude sequence-dependent backbone fluctuations might flag the genome for specific DNA recognition.

Probing transient Hoogsteen hydrogen bonds in canonical duplex DNA using NMR relaxation dispersion and single-atom substitution.

Nucleic acids transiently morph into alternative conformations that can be difficult to characterize at the atomic level by conventional methods because they exist for too little time and in too little abundance. We recently reported evidence for transient Hoogsteen (HG) base pairs in canonical B-DNA based on NMR carbon relaxation dispersion. While the carbon chemical shifts measured for the transient state were consistent with a syn orientation for the purine base, as expected for A(syn)•T(anti) and G(syn)•C(+)(anti) HG base pairing, HG type hydrogen bonding could only be inferred indirectly. Here, we develop two independent approaches for directly probing transient changes in N-H···N hydrogen bonds and apply them to the characterization of transient Hoogsteen type hydrogen bonds in canonical duplex DNA. The first approach takes advantage of the strong dependence of the imino nitrogen chemical shift on hydrogen bonding and involves measurement of R(1ρ) relaxation dispersion for the hydrogen-bond donor imino nitrogens in G and T residues. In the second approach, we assess the consequence of substituting the hydrogen-bond acceptor nitrogen (N7) with a carbon (C7H7) on both carbon and nitrogen relaxation dispersion data. Together, these data allow us to obtain direct evidence for transient Hoogsteen base pairs that are stabilized by N-H···N type hydrogen bonds in canonical duplex DNA. The methods introduced here greatly expand the utility of NMR in the structural characterization of transient states in nucleic acids.

Thermodynamics of RNA melting, one base pair at a time.

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe "microscopic" enthalpy-entropy compensation--often seen "macroscopically" across a series of related molecular species--across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy-entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.

Intrinsic dynamics of DNA-polymer complexes: a mechanism for DNA release.

The transfer of genetic material into cells using nonviral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, line width, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR time scale (

Extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon R(1rho) NMR spectroscopy.

We present an off-resonance carbon R(1rho) NMR experiment utilizing weak radiofrequency fields and selective polarization transfers for quantifying chemical-exchange processes in nucleic acids. The experiment extends the range of accessible time scales to approximately 10 ms, and its time-saving feature makes it possible to thoroughly map out dispersion profiles and conduct measurements at natural abundance. The experiment unveiled microsecond-to-millisecond exchange dynamics in a uniformly labeled A-site rRNA and in unlabeled, damaged DNA that would otherwise be difficult to characterize by conventional methods.

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR.

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific (13)C/(15)N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs.

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

Widespread transient Hoogsteen base pairs in canonical duplex DNA with variable energetics.

Hoogsteen (HG) base pairing involves a 180° rotation of the purine base relative to Watson-Crick (WC) base pairing within DNA duplexes, creating alternative DNA conformations that can play roles in recognition, damage induction and replication. Here, using nuclear magnetic resonance R1ρ relaxation dispersion, we show that transient HG base pairs occur across more diverse sequence and positional contexts than previously anticipated. We observe sequence-specific variations in HG base pair energetic stabilities that are comparable with variations in WC base pair stability, with HG base pairs being more abundant for energetically less favourable WC base pairs. Our results suggest that the variations in HG stabilities and rates of formation are dominated by variations in WC base pair stability, suggesting a late transition state for the WC-to-HG conformational switch. The occurrence of sequence and position-dependent HG base pairs provide a new potential mechanism for achieving sequence-dependent DNA transactions.

Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis.

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.