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Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semi-rigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically-relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semi-rigid linker provides enough molecular spacing to allow a single monomer to fully stack with base pairs, comparable to the monomeric parent ruthenium complex. The DNA base pairs in the equilibrium threading intercalated state are likely intact and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex., (Copyright © 2025 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

It is widely appreciated that double stranded DNA (dsDNA) is subjected to strong and dynamic mechanical forces in cells. Under increasing tension B-DNA, the most stable double-stranded (ds) form of DNA, undergoes cooperative elongation into a mixture of S-DNA and single stranded DNA (ssDNA). Despite significant effort, the structure, energetics, kinetics and the biological role of S-DNA remains obscure. We here stretch 60 base pair (bp) dsDNA oligonucleotides with a variable number of tricyclic cytosine, tC, modifications using optical tweezers. We observe multiple fast cooperative and reversible two-state transitions between B-DNA and S-DNA. Notably, tC modifications increase the transition force, while reducing the transition extension and free energy due to progressively increasing fraying of the dsDNA ends. We quantify the average number of bps undergoing the B-to-S transition, as well as the free energies and rates. This allows us to reconstruct the B-to-S free energy profiles in absence of force. We conclude that S-DNA is an entirely force-induced state, and that the B-to-S transition is much faster than internal dsDNA melting. We hypothesize that S-DNA may have a role as a transient intermediate in, for example, molecular motor-induced local dsDNA strand separation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

LINE-1 (L1) is a parasitic retrotransposable DNA element, active in primates for the last 80-120 Myr. L1 has generated nearly one-third of the human genome by copying its transcripts, and those of other genetic elements (e.g. Alu and SVA), into genomic DNA by target site-primed reverse transcription (TPRT) and remains active in modern humans. L1 encodes two proteins that bind their encoding transcript (cis preference) to form an L1 ribonucleoprotein (RNP) that mediates retrotransposition. ORF2p provides reverse transcriptase and endonuclease activity. ORF1p, its major component, is a homo-trimeric phospho-protein that binds single-stranded nucleic acid (ssNA) with high affinity and exhibits nucleic acid (NA) chaperone activity. We used optical tweezers to examine ORF1p binding to individual single-stranded DNA (ssDNA) molecules and found that the arrangement of ORF1p on the ssDNA depends on their molar ratio. When the concentration of ORF1p is just sufficient to saturate the entire NA molecule, the nucleoprotein (NP) is compact and stable. However, additional ORF1p binds and destabilizes the compacted NP, allowing it to engage a second ssDNA. Our results suggest that ORF1p displaced from its RNA template during TPRT could bind and destabilize remaining downstream L1 RNP, making them susceptible to hijacking by non-L1 templates, and thereby enable retrotransposition of non-L1 transcripts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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 K D ~ 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.

The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA binding to component histones of the nucleosome under a variety of conditions are essential to understand these processes at the molecular level. To accomplish this, we employ three specific single-molecule techniques: force disruption (FD) with optical tweezers, confocal imaging (CI) in a combined fluorescence plus optical trap, and survival probability (SP) measurements of disrupted and reformed nucleosomes. Short arrays of positioned nucleosomes serve as a template for study, facilitating rapid quantification of kinetic parameters. These arrays are then exposed to FACT (FAcilitates Chromatin Transcription), a non-ATP-driven heterodimeric nuclear chaperone known to both disrupt and tether histones during transcription. FACT binding drives off the outer wrap of DNA and destabilizes the histone-DNA interactions of the inner wrap as well. This reorganization is driven by two key domains with distinct function. FD experiments show the SPT16 MD domain stabilizes DNA-histone contacts, while the HMGB box of SSRP1 binds DNA, destabilizing the nucleosome. Surprisingly, CI experiments do not show tethering of disrupted histones, but increased rates of histone release from the DNA. SI experiments resolve this, showing that the two active domains of FACT combine to chaperone nucleosome reassembly after the timely release of force. These combinations of single-molecule approaches show FACT is a true nucleosome catalyst, lowering the barrier to both disruption and reformation., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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) 4 Ru 2 ] 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) 4 Ru 2 ] 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., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

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 ~10 4 . Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion.

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.

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.

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.

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.

Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

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.

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein plays an essential role in several stages of HIV-1 replication. One important function of HIV-1 NC is to act as a nucleic acid chaperone, in which the protein facilitates nucleic acid rearrangements important for reverse transcription and recombination. NC contains only 55 amino acids, with 15 basic residues and two zinc fingers, each having a single aromatic residue (Phe16 and Trp37). Despite its simple structure, HIV-1 NC appears to have optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid nucleic acid annealing. Here we combine single molecule DNA stretching experiments with ensemble solution studies of protein-nucleic acid binding affinity, oligonucleotide annealing, and nucleic acid aggregation to measure the characteristics of wild-type (WT) and aromatic residue mutants of HIV-1 NC that are important for nucleic acid chaperone activity. These in vitro results are compared to in vivo HIV-1 replication for viruses containing the same mutations. This work allows us to directly relate HIV-1 NC structure with its function as a nucleic acid chaperone in vitro and in vivo. We show that replacement of either aromatic residue with another aromatic residue results in a protein that strongly resembles WT NC. In contrast, single amino acid substitutions of either Phe16Ala or Trp37Ala significantly slow down NC's DNA interaction kinetics, while retaining some helix-destabilization capability. A double Phe16Ala/Trp37Ala substitution further reduces the latter activity. Surprisingly, the ensemble nucleic acid binding, annealing, and aggregation properties are not significantly altered for any mutant except the double aromatic substitution with Ala. Thus, elimination of a single aromatic residue from either zinc finger strongly reduces NC's chaperone activity as determined by single molecule DNA stretching experiments without significantly altering its ensemble-averaged biochemical properties. Importantly, the substitution of aromatic residues with Ala progressively decreases NC's nucleic acid chaperone activity while also progressively inhibiting viral replication. Taken together, these data support the critical role of HIV-1 NC's aromatic residues, and establish a direct and statistically significant correlation between nucleic acid chaperone activity and viral replication., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

HMGB (high-mobility group box) proteins are members of a class of small proteins that are ubiquitous in eukaryotic cells and nonspecifically bind to DNA, inducing large-angle DNA bends, enhancing the flexibility of DNA, and likely facilitating numerous important biological interactions. To determine the nature of this behavior for different HMGB proteins, we used atomic force microscopy to quantitatively characterize the bend angle distributions of DNA complexes with human HMGB2(Box A), yeast Nhp6A, and two chimeric mutants of these proteins. While all of the HMGB proteins bend DNA to preferred angles, Nhp6A promoted the formation of higher-order oligomer structures and induced a significantly broader distribution of angles, suggesting that the mechanism of Nhp6A is like a flexible hinge more than that of HMGB2(Box A). To determine the structural origins of this behavior, we used portions of the cationic N-terminus of Nhp6A to replace corresponding HMGB2(Box A) sequences. We found that the oligomerization and broader angle distribution correlated directly with the length of the N-terminus incorporated into the HMGB2(Box A) construct. Therefore, the basic N-terminus of Nhp6A is responsible for its ability to act as a flexible hinge and to form high-order structures., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

DNA-protein interactions may be observed on single molecules with a variety of techniques. However, quantifying the binding affinity is difficult and often requires many (∼100) individual events to characterize the interaction. We use a single λ DNA molecule that provides a lattice of binding sites for proteins. Extending and relaxing the tethered molecule reversibly melts DNA, allowing it to be converted between double-stranded (ds) and single-stranded (ss) forms. By monitoring changes in the properties of the DNA as a function of added protein concentration and fitting to binding models, the DNA-protein interaction may be characterized and quantified. As an example, the high mobility group protein HMGB1(box A  +  B) is observed to stabilize dsDNA. Measuring the strength of this effect allows us to determine the equilibrium association constant for HMGB1(box A  +  B) binding to dsDNA.

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function., ((c) 2010 Elsevier B.V. All rights reserved.)

Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double-stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double-stranded structure. Proteins binding to both double- and single-stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single-stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double-stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding., (2008 Wiley Periodicals, Inc.)

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.

The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

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.

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.

High mobility group (HMG) proteins are nuclear proteins believed to significantly affect DNA interactions by altering nucleic acid flexibility. Group B (HMGB) proteins contain HMG box domains known to bind to the DNA minor groove without sequence specificity, slightly intercalating base pairs and inducing a strong bend in the DNA helical axis. A dual-beam optical tweezers system is used to extend double-stranded DNA (dsDNA) in the absence as well as presence of a single box derivative of human HMGB2 [HMGB2(box A)] and a double box derivative of rat HMGB1 [HMGB1(box A+box B)]. The single box domain is observed to reduce the persistence length of the double helix, generating sharp DNA bends with an average bending angle of 99+/-9 degrees and, at very high concentrations, stabilizing dsDNA against denaturation. The double box protein contains two consecutive HMG box domains joined by a flexible tether. This protein also reduces the DNA persistence length, induces an average bending angle of 77+/-7 degrees , and stabilizes dsDNA at significantly lower concentrations. These results suggest that single and double box proteins increase DNA flexibility and stability, albeit both effects are achieved at much lower protein concentrations for the double box. In addition, at low concentrations, the single box protein can alter DNA flexibility without stabilizing dsDNA, whereas stabilization at higher concentrations is likely achieved through a cooperative binding mode.

We used single DNA molecule stretching to investigate DNA intercalation by ethidium and three ruthenium complexes. By measuring ligand-induced DNA elongation at different ligand concentrations, we determined the binding constant and site size as a function of force. Both quantities depend strongly on force and, in the limit of zero force, converge to the known bulk solution values, when available. This approach allowed us to distinguish the intercalative mode of ligand binding from other binding modes and allowed characterization of intercalation with binding constants ranging over almost six orders of magnitude, including ligands that do not intercalate under experimentally accessible solution conditions. As ligand concentration increased, the DNA stretching curves saturated at the maximum amount of ligand intercalation. The results showed that the applied force partially relieves normal intercalation constraints. We also characterized the flexibility of intercalator-saturated dsDNA for the first time.

The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments., ((c) 2006 Wiley Periodicals, Inc.)

When a single DNA molecule is stretched beyond its normal contour length, a force-induced melting transition is observed. Ethidium binding increases the DNA contour length, decreases the elongation upon melting, and increases the DNA melting force in a manner that is consistent with the ethidium-induced changes in duplex DNA stability known from thermal melting studies. The DNA stretching curves map out a phase diagram and critical point in the force-extension-ethidium concentration space. Intercalation occurs between alternate base pairs at low forces and between every base pair at high forces.

The inclusion of family members in employer-sponsored health promotion activities is an area gaining attention. Several investigations into the state-of-the-art of employer-sponsored health promotion programs for family members have been undertaken. This chapter describes the findings of the Washington Business Group on Health project. The costs and benefits of family-related health care are discussed. The authors also outline a number of ways for companies to recruit family members.

Seventy-five men undergoing vasectomy were tested by radial immunodiffusion assay prevasectomy and at 6 weeks and 3, 6, and 12 months postvasectomy for possible changes in serum immunoglobulin (Ig) levels. No significant changes occurred when the results were analyzed on a group basis per time period. However, when each individual's prevasectomy Ig level was considered as his norm, significant changes appeared to occur in serum IgG levels at 6 weeks, 6 months, and 12 months postvasectomy; in serum IgM levels at 6 weeks; but not, to date, in serum IgA levels. Seasonal, environmental, and infectious factors do not seem to be associated with the changes found. No significant seminal Ig changes have been demonstrated. Consideration of these findings and implications for possible immunopathology are briefly discussed.

Bacillus megaterium strain ATCC 19213 secretes a ferric-chelating secondary hydroxamic acid, whereas a mutant (strain SK11) derived from it cannot produce a hydroxamate. Strain SK11 could be cultivated in a sucrose-mineral salts medium (treated with Chelex 100 to reduce trace metals) in the absence of added hydroxamate, if the inoculum was high. The lowest iron supplements necessary for maximal growth of both strains were equivalent (0.01 to 0.04 mug of iron per ml). Addition of either aluminum (0.5 mug/ml) or chromium (0.1 mug/ml) to the medium prevented full growth of strain SK11 at the minimal iron concentration, although elevated iron (1 mug/ml) reversed this inhibition. The iron-free secondary hydroxamate, Desferal, also abolished aluminum and chromium inhibition of strain SK11, producing maximal population densities at the low iron concentration. Growth of the hydroxamate-producing strain 19213 was not altered significantly by the aluminum or chromium levels which inhibited strain SK11. However, strain 19213 responded to these metals by increasing its secretion of a secondary hydroxamate. It was concluded that aluminum and chromium interfered with iron incorporation, either directly or by formation of nonutilizable aggregates with iron. The secondary hydroxamates may have overcome this interference by solubilization of iron for delivery to a single uptake process, or the ferric-hydroxamate chelate may enter the cell by an alternate route.