How environmental solution conditions determine the compaction velocity of single DNA molecules.
ABSTRACT: Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg(2+) for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem ?-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA-protein interactions in vivo.
Project description:Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of adenosine triphosphate hydrolysis-dependent translocation along double-stranded DNA. Condensin's translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ?10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.
Project description:BACKGROUND AND PURPOSE:Locally compacting the mesh of a flow diverter by a dynamic push-pull technique can accelerate intracranial aneurysm healing. We asked how this deployment strategy compares with overlapping 2 flow diverters for aneurysmal flow reduction. MATERIALS AND METHODS:Using a high-fidelity virtual stent placement method, we simulated 3 flow-diverter strategies (single noncompacted, 2 overlapped, and single compacted) in 3 aneurysms (fusiform, large saccular, and medium saccular). Computational fluid dynamics analysis provided posttreatment hemodynamic parameters, including time-averaged inflow rate, aneurysm-averaged velocity, wall shear stress, total absolute circulation, and turnover time. We examined the relationship between the achieved degree of compaction and aneurysm orifice area. RESULTS:Flow-diverter compaction resulted in a compaction coverage of 57%, 47%, and 22% over the orifice of the fusiform, large, and medium saccular aneurysm, respectively. Compaction coverage increased linearly with orifice area. In the fusiform aneurysm, the single compacted flow diverter accomplished more aneurysmal flow reduction than the other 2 strategies, as indicated by all 5 hemodynamic parameters. In the 2 saccular aneurysms, the overlapped flow diverters achieved the most flow reduction, followed by the single compacted and the noncompacted flow diverter. CONCLUSIONS:Compacting a single flow diverter can outperform overlapping 2 flow diverters in aneurysmal flow reduction, provided that the compaction produces a mesh denser than 2 overlapped flow diverters and this denser mesh covers a sufficient portion of the aneurysm orifice area, for which we suggest a minimum of 50%. This strategy is most effective for aneurysms with large orifices, especially fusiform aneurysms.
Project description:The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria.
Project description:Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK(30)), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH(12)K(18). Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH(12)K(18) to levels comparable with branched polyethyleneimine. PEG-CH(12)K(18) compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK(30) DNA nanoparticles. PEG-CH(12)K(18) DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH(12)K(18) DNA nanoparticles improved the in vitro gene transfer by ~20-fold over PEG-CK(30) DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.
Project description:Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.
Project description:Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK(30)PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK(30)PEG DNPs made using two different molecular weights of PEG, CK(30)PEG(10k) and CK(30)PEG(5k). We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2h, 37±19% and 47±8% for CK(30)PEG(10k) and CK(30)PEG(5k), respectively. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs moved within live cells at average velocities of 0.09±0.04?m/s and 0.11±0.04?m/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.
Project description:Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered ? DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea.IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.
Project description:Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.
Project description:Nuclei in dry Arabidopsis thaliana seeds are very small and have highly condensed chromatin. Nuclear shrinkage and chromatin compaction occur during seed maturation and have been shown to be independent, developmentally controlled processes. To confirm this genetically, we studied chromatin compaction in a mutant of the seed developmental regulator ABA INSENSITIVE 3, and in a double mutant of the nuclear matrix proteins LITTLE NUCLEI 1 and 2. Our results indicated that the nuclear shrinking and chromatin condensation during seed maturation can be genetically uncoupled, confirming that these are independent processes. In addition, we demonstrated that transcript levels of siliques toward the end of seed maturation are comparable to those in vegetative tissues, despite the highly compacted chromatin, small nuclear volume and low hydration status of seeds.
Project description:Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones.Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin.These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.