Mechanism of DNA Intercalation by Chloroquine Provides Insights into Toxicity.

Chloroquine has been used as a potent antimalarial, anticancer drug, and prophylactic. While chloroquine is known to interact with DNA, the details of DNA-ligand interactions have remained unclear. Here we characterize chloroquine-double-stranded DNA binding with four complementary approaches, including optical tweezers, atomic force microscopy, duplex DNA melting measurements, and isothermal titration calorimetry. We show that chloroquine intercalates into double stranded DNA (dsDNA) with a KD ~ 200 µM, and this binding is entropically driven. We propose that chloroquine-induced dsDNA intercalation, which happens in the same concentration range as its observed toxic effects on cells, is responsible for the drug's cytotoxicity.

Left versus right: Exploring the effects of chiral threading intercalators using optical tweezers.

Small-molecule DNA-binding drugs have shown promising results in clinical use against many types of cancer. Understanding the molecular mechanisms of DNA binding for such small molecules can be critical in advancing future drug designs. We have been exploring the interactions of ruthenium-based small molecules and their DNA-binding properties that are highly relevant in the development of novel metal-based drugs. Previously we have studied the effects of the right-handed binuclear ruthenium threading intercalator ΔΔ-[µ-bidppz(phen)4Ru2]4+, or ΔΔ-P for short, which showed extremely slow kinetics and high-affinity binding to DNA. Here we investigate the left-handed enantiomer ΛΛ-[µ-bidppz(phen)4Ru2]4+, or ΛΛ-P for short, to study the effects of chirality on DNA threading intercalation. We employ single-molecule optical trapping experiments to understand the molecular mechanisms and nanoscale structural changes that occur during DNA binding and unbinding as well as the association and dissociation rates. Despite the similar threading intercalation binding mode of the two enantiomers, our data show that the left-handed ΛΛ-P complex requires increased lengthening of the DNA to thread, and it extends the DNA more than double the length at equilibrium compared with the right-handed ΔΔ-P. We also observed that the left-handed ΛΛ-P complex unthreads three times faster than ΔΔ-P. These results, along with a weaker binding affinity estimated for ΛΛ-P, suggest a preference in DNA binding to the chiral enantiomer having the same right-handed chirality as the DNA molecule, regardless of their common intercalating moiety. This comparison provides a better understanding of how chirality affects binding to DNA and may contribute to the development of enhanced potential cancer treatment drug designs.

Single and double box HMGB proteins differentially destabilize nucleosomes.

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

Quantifying the stability of oxidatively damaged DNA by single-molecule DNA stretching.

One of the most common DNA lesions is created when reactive oxygen alters guanine. 8-oxo-guanine may bind in the anti-conformation with an opposing cytosine or in the syn-conformation with an opposing adenine paired by transversion, and both conformations may alter DNA stability. Here we use optical tweezers to measure the stability of DNA hairpins containing 8-oxoguanine (8oxoG) lesions, comparing the results to predictive models of base-pair energies in the absence of the lesion. Contrasted with either a canonical guanine-cytosine or adenine-thymine pair, an 8oxoG-cytosine base pair shows significant destabilization of several kBT. The magnitude of destabilization is comparable to guanine-thymine 'wobble' and cytosine-thymine mismatches. Furthermore, the measured energy of 8oxoG-adenine corresponds to theoretical predictions for guanine-adenine pairs, indicating that oxidative damage does not further destabilize this mismatch in our experiments, in contrast to some previous observations. These results support the hypothesis that oxidative damage to guanine subtly alters the direction of the guanine dipole, base stacking interactions, the local backbone conformation, and the hydration of the modified base. This localized destabilization under stress provides additional support for proposed mechanisms of enzyme repair.

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA.

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [µ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin.

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein-protein interactions.

Differential contribution of basic residues to HIV-1 nucleocapsid protein's nucleic acid chaperone function and retroviral replication.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC's NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC's aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.

Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility.

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (~10 s(-1)) are higher than those observed for wild-type proteins (~0.1-1.0 s(-1)), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB-DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

Mechanical properties of base-modified DNA are not strictly determined by base stacking or electrostatic interactions.

This work probes the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. Here we explore the relationship between base stacking, functional group occupancy of the DNA minor and major grooves, and DNA mechanical properties. We study double-helical DNA molecules substituting either inosine for guanosine or 2,6-diaminopurine for adenine. These DNA variants, respectively, remove or add an amino group from the DNA minor groove, with corresponding changes in hydrogen-bonding and base stacking energy. Using the techniques of ligase-catalyzed cyclization kinetics, atomic force microscopy, and force spectroscopy with optical tweezers, we show that these DNA variants have bending persistence lengths within the range of values reported for sequence-dependent variation of the natural DNA bases. Comparison with seven additional DNA variants that modify the DNA major groove reveals that DNA bending stiffness is not correlated with base stacking energy or groove occupancy. Data from circular dichroism spectroscopy indicate that base analog substitution can alter DNA helical geometry, suggesting a complex relationship among base stacking, groove occupancy, helical structure, and DNA bend stiffness.

Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D.

Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (≈ 10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.

Cationic Residues of the HIV-1 Nucleocapsid Protein Enable DNA Condensation to Maintain Viral Core Particle Stability during Reverse Transcription.

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.

HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating.

Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes.

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.

Mechanism of DNA flexibility enhancement by HMGB proteins.

The mechanism by which sequence non-specific DNA-binding proteins enhance DNA flexibility is studied by examining complexes of double-stranded DNA with the high mobility group type B proteins HMGB2 (Box A) and HMGB1 (Box A+B) using atomic force microscopy. DNA end-to-end distances and local DNA bend angle distributions are analyzed for protein complexes deposited on a mica surface. For HMGB2 (Box A) binding we find a mean induced DNA bend angle of 78 degrees, with a standard error of 1.3 degrees and a SD of 23 degrees, while HMGB1 (Box A+B) binding gives a mean bend angle of 67 degrees, with a standard error of 1.3 degrees and a SD of 21 degrees. These results are consistent with analysis of the observed global persistence length changes derived from end-to-end distance measurements, and with results of DNA-stretching experiments. The moderately broad distributions of bend angles induced by both proteins are inconsistent with either a static kink model, or a purely flexible hinge model for DNA distortion by protein binding. Therefore, the mechanism by which HMGB proteins enhance the flexibility of DNA must differ from that of the Escherichia coli HU protein, which in previous studies showed a flat angle distribution consistent with a flexible hinge model.

Specific Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein Deduced from Hairpin Unfolding.

RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide transactivation response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. Yet it has remained difficult to elucidate the way NC targets and dramatically destabilizes this hairpin while only weakly affecting the annealed product. In this work, we used optical tweezers to measure the stability of TAR and found that adding NC destabilized the hairpin and simultaneously caused a distinct change in both the height and location of the energy barrier. This data was matched to an energy landscape predicted from a simple theory of definite base pair destabilization. Comparisons revealed the specific binding sites found by NC along the irregular TAR hairpin. Furthermore, specific binding explained both the unusual shift in the transition state and the much weaker effect on the annealed product. These experiments illustrate a general method of energy landscape transformation that exposes important physical insights.

Significant Differences in RNA Structure Destabilization by HIV-1 GagDp6 and NCp7 Proteins.

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and GagDp6 destabilize the TAR hairpin, GagDp6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike GagDp6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion.

DNA overstretching in the presence of glyoxal: structural evidence of force-induced DNA melting.

When a long DNA molecule is stretched beyond its B-form contour length, a transition occurs in which its length increases by a factor of 1.7, with very little force increase. A quantitative model was proposed to describe this transition as force-induced melting, where double-stranded DNA is converted into single-stranded DNA. The force-induced melting model accurately describes the thermodynamics of DNA overstretching as a function of solution conditions and in the presence of DNA binding ligands. An alternative explanation suggests a transformation into S-DNA, a double-stranded form which preserves the interstrand base pairing. To determine the extent to which DNA base pairs are exposed to solution during the transition, we held DNA overstretched to different lengths within the transition in the presence of glyoxal. If overstretching involved strand separation, then force-melted basepairs would be glyoxal-modified, thus essentially permanently single-stranded. Subsequent stretches confirm that a significant fraction of the DNA melted by force is permanently melted. This result demonstrates that DNA overstretching is accompanied by a disruption of the DNA helical structure, including a loss of hydrogen bonding.

Mechanically manipulating the DNA threading intercalation rate.

The dumbbell shaped binuclear ruthenium complex DeltaDelta-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur.

Constructing Free Energy Landscapes of Nucleic Acid Hairpin Unfolding.

Single nucleic acid molecules form hairpins that may stabilize secondary and tertiary structures as well as perform enzymatic and other chemical functions. Considerable progress has been made in the effort to understand the contributions of various factors to the stability of a given hairpin sequence. For a given sequence, it is possible to compute both the most likely structural arrangements and their associated free energies over a range of experimental conditions. However, there are many observed hairpin irregularities for which the energies and function are not well understood. Here we examine the irregular RNA Transactivation Response (TAR) hairpin from the HIV-1 genome. Using single molecule optical tweezers, the hairpin is force unfolded, revealing the overall unfolding free energy and the character of the transition state. These measurements allow the construction of a simple energy landscape from unfolding measurements, which can be directly compared to a theoretical landscape. This method is easily adapted to other structures, including the effects of noncanonical bases and even ligand binding.