Nanoscale Interaction of Endonuclease APE1 with DNA.

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions.

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

Beyond Sequence: Internucleosomal Interactions Dominate Array Assembly.

The organization of the nucleosome array is a critical component of the chromatin assembly into higher order structure as well as its function. Here, we investigated the contributions of the DNA sequence and internucleosomal interactions on the organization of the nucleosomal arrays in compact structures using atomic force microscopy. We assembled nucleosomes on DNA substrates allowing for the formation of tetranucleosomes. We found that nucleosomes are capable of close positioning with no discernible space between them, even in the case of assembled dinucleosomes. This morphology of the array is in contrast with that observed for arrays assembled with repeats of the nucleosome positioning motifs separated by uniform spacers. Simulated assembly of tetranucleosomes by random placement along the substrates revealed that nucleosome array compaction is promoted by the interaction of the nucleosomes. We developed a theoretical model to account for the role of DNA sequence and internucleosomal interactions in the formation of the nucleosome structures. These findings suggest that, in the chromatin assembly, the affinity of the nucleosomes to the DNA sequence and the strengths of the internucleosomal interactions are the two major factors defining the compactness of the chromatin.

Nanorings to Probe Mechanical Stress of Single-Stranded DNA Mediated by the DNA Duplex.

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins.

Free Cholesterol Accelerates Aß Self-Assembly on Membranes at Physiological Concentration.

The effects of membranes on the early-stage aggregation of amyloid ß (Aß) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer's disease. We have shown that direct Aß-membrane interactions dramatically enhance the Aß aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aß monomers and significantly changes the conformational sampling of Aß monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aß-lipid interaction is an important factor in the disease prone amyloid assembly process.

Hybrid resolution molecular dynamics simulations of amyloid proteins interacting with membranes.

A broad range of human diseases, including Alzheimer's and Parkinson's diseases, arise from or have as key players intrinsically disordered proteins. The aggregation of these amyloid proteins into fibrillar aggregates are the key events of such diseases. Characterizing the conformation dynamics of the proteins involved is crucial for understanding the molecular mechanisms of aggregation, which in turn is important for drug development efforts against these diseases. Computational approaches have provided extensive detail about some steps of the aggregation process, however the biologically relevant elements responsible for the aggregation and or aggregation propagation have not been fully characterized. Here we describe a hybrid resolution molecular dynamics simulation method that can be employed to investigate the interaction of amyloid proteins with lipid membranes, shown to dramatically accelerate the aggregation propensity of amyloid proteins. The hybrid resolution method enables routine and accurate simulation of multi-protein and complex membrane systems, mimicking biologically relevant lipid membranes, on microsecond time scales. The hybrid resolution method was applied to computer modeling of the interactions of α -synuclein protein with a mixed lipid bilayer.

Restriction of RecG translocation by DNA mispairing.

BACKGROUND: The RecG DNA helicase plays a crucial role in stalled replication fork rescue. We have recently discovered that interaction of RecG with single-strand DNA binding protein (SSB) remodels RecG, allowing it to spontaneously translocate upstream of the fork. Based on these findings, we hypothesized that mispairing of DNA could limit such translocation of RecG. METHODS: Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. RESULTS: We found that a CC mispairing, at a distance of 30 bp from the fork position, prevents translocation of RecG over this mispairing. A G-bulge, placed at the same distance, also has a similar blocking efficiency. However, a CC mispairing, 10 bp away from the fork, does not prevent RecG translocation beyond 10 bp distance, but decreases complex yield. Modeling of RecG-DNA complexes show that 10 bp distance from the fork is within the binding footprint of RecG on DNA. CONCLUSIONS: Our results suggest that the RecG translocation upstream of the replication fork is limited by mispairings in the parental arm of the replication fork. General significance These findings led us to propose dual functions for RecG, in which the thermally driven translocation of RecG can be a mechanism for the additional control of the DNA paring in which RecG can detect the lesions in front of the replication fork, adding to the fidelity of the DNA replication machinery.

DNA Looping Mediated by Site-Specific SfiI-DNA Interactions.

Interactions between distant DNA segments play important roles in various biological processes, such as DNA recombination. Certain restriction enzymes create DNA loops when two sites are held together and then cleave the DNA. DNA looping is important during DNA synapsis. Here we investigated the mechanisms of DNA looping by restriction enzyme SfiI by measuring the properties of the system at various temperatures. Different sized loop complexes, mediated by SfiI-DNA interactions, were visualized with AFM. The experimental results revealed that small loops are more favorable compared to other loop sizes at all temperatures. Our theoretical model found that entropic cost dominates at all conditions, which explains the preference for short loops. Furthermore, specific loop sizes were predicted as favorable from an energetic point of view. These predictions were tested by experiments with transiently assembled SfiI loops on a substrate with a single SfiI site.

Cholesterol in Membranes Facilitates Aggregation of Amyloid ß Protein at Physiologically Relevant Concentrations.

The formation of amyloid ß (1-42) (Aß42) oligomers is considered to be a critical step in the development of Alzheimer's disease (AD). However, the mechanism underlying this process at physiologically low concentrations of Aß42 remains unclear. We have previously shown that oligomers assemble at such low Aß42 monomer concentrations in vitro on phospholipid membranes. We hypothesized that membrane composition is the factor controlling the aggregation process. Accumulation of cholesterol in membranes is associated with AD development, suggesting that insertion of cholesterol into membranes may initiate the Aß42 aggregation, regardless of a low monomer concentration. We used atomic force microscopy (AFM) to test the hypothesis and directly visualize the aggregation process of Aß42 on the surface of a lipid bilayer depending on the cholesterol presence. Time-lapse AFM imaging unambiguously demonstrates that cholesterol in the lipid bilayer significantly enhances the aggregation process of Aß42 at nanomolar monomer concentration. Quantitative analysis of the AFM data shows that both the number of Aß42 oligomers and their sizes grow when cholesterol is present. Importantly, the aggregation process is dynamic, so the aggregates assembled on the membrane can dissociate from the bilayer surface into the bulk solution. Computational modeling demonstrated that the lipid bilayer containing cholesterol had an elevated affinity to Aß42. Moreover, monomers adopted the aggregation-prone conformations present in amyloid fibrils. The results lead to the model for the on-surface aggregation process in which the self-assembly of Aß oligomers is controlled by the lipid composition of cellular membranes.

Site-Search Process for Synaptic Protein-DNA Complexes.

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.

Interaction of Aß42 with Membranes Triggers the Self-Assembly into Oligomers.

The self-assembly of amyloid ß (Aß) proteins into oligomers is the major pathogenic event leading to Alzheimer's disease (AD). Typical in vitro experiments require high protein concentrations, whereas the physiological concentration of Aß is in the picomolar to low nanomolar range. This complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here, we demonstrate that Aß42 self-assembles into aggregates on membrane bilayers at low nanomolar concentrations - a pathway in which the membrane plays the role of a catalyst. Additionally, physiological ionic conditions (150 mM NaCl) significantly enhance on-membrane aggregation, leading to the rapid formation of oligomers. The self-assembly process is reversible, so assembled aggregates can dissociate from the membrane surface into the bulk solution to further participate in the aggregation process. Molecular dynamics simulations demonstrate that the transient membrane-Aß interaction dramatically changes the protein conformation, facilitating the assembly of dimers. The results indicate peptide-membrane interaction is the critical step towards oligomer formation at physiologically low protein concentrations.

Assembly of α-synuclein aggregates on phospholipid bilayers.

The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.

Spontaneous self-assembly of amyloid ß (1-40) into dimers.

The self-assembly and fibrillation of amyloid ß (Aß) proteins is the neuropathological hallmark of Alzheimer's disease. However, the molecular mechanism of how disordered monomers assemble into aggregates remains largely unknown. In this work, we characterize the assembly of Aß (1-40) monomers into dimers using long-time molecular dynamics simulations. Upon interaction, the monomers undergo conformational transitions, accompanied by change of the structure, leading to the formation of a stable dimer. The dimers are stabilized by interactions in the N-terminal region (residues 5-12), in the central hydrophobic region (residues 16-23), and in the C-terminal region (residues 30-40); with inter-peptide interactions focused around the N- and C-termini. The dimers do not contain long ß-strands that are usually found in fibrils.

High-speed atomic force microscopy reveals structural dynamics of α-synuclein monomers and dimers.

α-Synuclein (α-syn) is the major component of the intraneuronal inclusions called Lewy bodies, which are the pathological hallmark of Parkinson's disease. α-Syn is capable of self-assembly into many different species, such as soluble oligomers and fibrils. Even though attempts to resolve the structures of the protein have been made, detailed understanding about the structures and their relationship with the different aggregation steps is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. Here we report the structural flexibility of α-syn monomers and dimers in an aqueous solution environment as probed by single-molecule time-lapse high-speed AFM. In addition, we present the molecular basis for the structural transitions using discrete molecular dynamics (DMD) simulations. α-Syn monomers assume a globular conformation, which is capable of forming tail-like protrusions over dozens of seconds. Importantly, a globular monomer can adopt fully extended conformations. Dimers, on the other hand, are less dynamic and show a dumbbell conformation that experiences morphological changes over time. DMD simulations revealed that the α-syn monomer consists of several tightly packed small helices. The tail-like protrusions are also helical with a small ß-sheet, acting as a "hinge". Monomers within dimers have a large interfacial interaction area and are stabilized by interactions in the non-amyloid central (NAC) regions. Furthermore, the dimer NAC-region of each α-syn monomer forms a ß-rich segment. Moreover, NAC-regions are located in the hydrophobic core of the dimer.

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks.

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of Escherichia coli RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks.

Nanoscale dynamics of centromere nucleosomes and the critical roles of CENP-A.

In the absence of a functioning centromere, chromosome segregation becomes aberrant, leading to an increased rate of aneuploidy. The highly specific recognition of centromeres by kinetochores suggests that specific structural characteristics define this region, however, the structural details and mechanism underlying this recognition remains a matter of intense investigation. To address this, high-speed atomic force microscopy was used for direct visualization of the spontaneous dynamics of CENP-A nucleosomes at the sub-second time scale. We report that CENP-A nucleosomes change conformation spontaneously and reversibly, utilizing two major pathways: unwrapping, and looping of the DNA; enabling core transfer between neighboring DNA substrates. Along with these nucleosome dynamics we observed that CENP-A stabilizes the histone core against dissociating to histone subunits upon unwrapping DNA, unique from H3 cores which are only capable of such plasticity in the presence of remodeling factors. These findings have implications for the dynamics and integrity of nucleosomes at the centromere.

Nano-assembly of amyloid ß peptide: role of the hairpin fold.

Structural investigations have revealed that ß hairpin structures are common features in amyloid fibrils, suggesting that these motifs play an important role in amyloid assembly. To test this hypothesis, we characterized the effect of the hairpin fold on the aggregation process using a model ß hairpin structure, consisting of two Aß(14-23) monomers connected by a turn forming YNGK peptide. AFM studies of the assembled aggregates revealed that the hairpin forms spherical structures whereas linear Aß(14-23) monomers form fibrils. Additionally, an equimolar mixture of the monomer and the hairpin assembles into non-fibrillar aggregates, demonstrating that the hairpin fold dramatically changes the morphology of assembled amyloid aggregates. To understand the molecular mechanism underlying the role of the hairpin fold on amyloid assembly, we performed single-molecule probing experiments to measure interactions between hairpin and monomer and two hairpin complexes. The studies reveal that the stability of hairpin-monomer complexes is much higher than hairpin-hairpin complexes. Molecular dynamics simulations revealed a novel intercalated complex for the hairpin and monomer and Monte Carlo modeling further demonstrated that such nano-assemblies have elevated stability compared with stability of the dimer formed by Aß(14-23) hairpin. The role of such folding on the amyloid assembly is also discussed.

A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces.

A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surface aggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surface aggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aß protein (Aß42), a decapeptide Aß(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surface aggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aß(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset.

Self-assembly of the full-length amyloid Aß42 protein in dimers.

The self-assembly of amyloid (Aß) proteins into nano-aggregates is a hallmark of Alzheimer's disease (AD) development, yet the mechanism of how disordered monomers assemble into aggregates remains elusive. Here, we applied long-time molecular dynamics simulations to fully characterize the assembly of Aß42 monomers into dimers. Monomers undergo conformational changes during their interaction, but the resulting dimer structures do not resemble those found in fibril structures. To identify natural conformations of dimers among a set of simulated ones, validation approaches were developed and applied, and a subset of dimer conformations were characterized. These dimers do not contain long ß-strands that are usually found in fibrils. The dimers are stabilized primarily by interactions within the central hydrophobic regions and the C-terminal regions, with a contribution from local hydrogen bonding. The dimers are dynamic, as evidenced by the existence of a set of conformations and by the quantitative analyses of the dimer dissociation process.

Aligned deposition and electrical measurements on single DNA molecules.

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)-poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg(2+) ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin-DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin-DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity.