HIV-1 uncoating requires long double-stranded reverse transcription products.

HIV-1 cores, which contain the viral genome and replication machinery, must disassemble (uncoat) during viral replication. However, the viral and host factors that trigger uncoating remain unidentified. Recent studies show that infectious cores enter the nucleus and uncoat near the site of integration. Here, we show that efficient uncoating of nuclear cores requires synthesis of a double-stranded DNA (dsDNA) genome >3.5 kb and that the efficiency of uncoating correlates with genome size. Core disruption by capsid inhibitors releases viral DNA, some of which integrates. However, most of the viral DNA is degraded, indicating that the intact core safeguards viral DNA. Atomic force microscopy and core content estimation reveal that synthesis of full-length genomic dsDNA induces substantial internal strain on the core to promote uncoating. We conclude that HIV-1 cores protect viral DNA from degradation by host factors and that synthesis of long double-stranded reverse transcription products is required to trigger efficient HIV-1 uncoating.

C-terminal Domain of T4 gene 32 Protein Enables Rapid Filament Reorganization and Dissociation.

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32's C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing.

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.

Dynamic structure of T4 gene 32 protein filaments facilitates rapid noncooperative protein dissociation.

Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32-ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32-ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.

Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates.

The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.

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.

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.

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.

The HIV-1 nucleocapsid chaperone protein forms locally compacted globules on long double-stranded DNA.

The nucleocapsid (NC) protein plays key roles in Human Immunodeficiency Virus 1 (HIV-1) replication, notably by condensing and protecting the viral RNA genome and by chaperoning its reverse transcription into double-stranded DNA (dsDNA). Recent findings suggest that integration of viral dsDNA into the host genome, and hence productive infection, is linked to a small subpopulation of viral complexes where reverse transcription was completed within the intact capsid. Therefore, the synthesized dsDNA has to be tightly compacted, most likely by NC, to prevent breaking of the capsid in these complexes. To investigate NC's ability to compact viral dsDNA, we here characterize the compaction of single dsDNA molecules under unsaturated NC binding conditions using nanofluidic channels. Compaction is shown to result from accumulation of NC at one or few compaction sites, which leads to small dsDNA condensates. NC preferentially initiates compaction at flexible regions along the dsDNA, such as AT-rich regions and DNA ends. Upon further NC binding, these condensates develop into a globular state containing the whole dsDNA molecule. These findings support NC's role in viral dsDNA compaction within the mature HIV-1 capsid and suggest a possible scenario for the gradual dsDNA decondensation upon capsid uncoating and NC loss.

Multiprotein E. coli SSB-ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions.

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB-ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.

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.

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.

HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunodeficiency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD's search function. These processes explain A3G's ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process.

Specific inter-domain interactions stabilize a compact HIV-1 Gag conformation.

HIV-1 Gag is a large multidomain poly-protein with flexible unstructured linkers connecting its globular subdomains. It is compact when in solution but assumes an extended conformation when assembled within the immature HIV-1 virion. Here, we use molecular dynamics (MD) simulations to quantitatively characterize the intra-domain interactions of HIV-1 Gag. We find that the matrix (MA) domain and the C-terminal subdomain CActd of the CA capsid domain can form a bound state. The bound state, which is held together primarily by interactions between complementary charged and polar residues, stabilizes the compact state of HIV-1 Gag. We calculate the depth of the attractive free energy potential between the MA/ CActd sites and find it to be about three times larger than the dimerization interaction between the CActd domains. Sequence analysis shows high conservation within the newly-found intra-Gag MA/CActd binding site, as well as its spatial proximity to other well known elements of Gag -such as CActd's SP1 helix region, its inositol hexaphosphate (IP6) binding site and major homology region (MHR), as well as the MA trimerization site. Our results point to a high, but yet undetermined, functional significance of the intra-Gag binding site. Recent biophysical experiments that address the binding specificity of Gag are interpreted in the context of the MA/CActd bound state, suggesting an important role in selective packaging of genomic RNA by Gag.

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.

RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids.

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.

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.

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.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.