Lipoplex thermodynamics: determination of DNA-cationic lipoid interaction energies

An experimental study of the cationic lipid-DNA binding affinity is presented. The binding free energy was determined by monitoring lipoplex dissociation under conditions of increasing salt concentration. The primary procedure was based on the extent of quenching by energy transfer of fluorophores on DNA molecules by fluorophore on a lipid as these molecules came into close association in the lipoplex. Titration calorimetry on the Dickerson dodecamer was also done, with results that were in agreement with the fluorescence data. Measurements on short oligonucleotides allowed estimation of the binding energy per nucleotide. The binding free energy is approximately 0.6 kcal/mole nucleotide for the Dickerson dodecamer and declines for longer oligonucleotides. The entropy gained upon complex formation is approximately 1 entropy unit per released counterion. The method was applied to long DNA molecules (herring and lambda-phage DNA) and revealed that complete dissociation occurs at 750 mM NaCl. Likely contributions of macromolecular desolvation and DNA flexibility to the binding energy are discussed.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Leukocyte Adhesion: What's the Catch?

A recent study shows that the leukocyte adhesion molecules known as selectins form 'catch' bonds, the dissociation rate of which decreases with increasing applied force. The ability of selectins to switch between catch and slip bonds, where dissociation increases with force, can explain the shear threshold effect, in which leukocyte adhesion goes through a maximum with increasing shear rate.     

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.     

Behavior of giant vesicles with anchored DNA molecules

We study changes in curvature and elastic properties of lipid membranes induced by anchoring of long hydrophilic polymers at low polymer surface concentrations (corresponding to the mushroom regime). The effect of anchored polymers on the membrane spontaneous curvature is characterized by monitoring the changes in the fluctuation spectra and the morphology of giant unilamellar vesicles. The polymers used in our study are fluorescently labeled and biotinylated lambda-phage DNA molecules which bind to biotinylated giant unilamellar vesicles via a biotin-avidin-biotin linkage. By varying the amount of biotinylated lipid in the membrane, we control the surface concentration of anchors. At low anchor concentrations, the spontaneous curvature of the membrane increases linearly with the DNA concentration. The linear increase is consistent with theoretical predictions for polymer surface concentrations in the mushroom regime. At higher anchor concentrations, which should still belong to the mushroom regime, the vesicles undergo budding transitions. In this latter regime, the bud size is used to estimate the polymer-induced membrane curvature.     

Nucleosome Assembly Depends on the Torsion in the DNA Molecule: A Magnetic Tweezers Study

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025-0.051 (most probably in the form of twist) stall the nucleosome assembly process.     

Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork.     

Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.     

Visualizing the assembly of human Rad51 filaments on double-stranded DNA

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these "molecular curtains" allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.     

Viral DNA sequences from incomplete particles of human adenovirus type 7

Large pools of empty viral capsids accumulate in cells infected by subgroup B human adenoviruses. Such infected cells also yield DNA-containing incomplete particles in larger quantities than cells infected with serotypes representing other adenovirus subgroups. DNA isolated from carefully purified classes of Ad7 incomplete particles was analyzed by restriction endonuclease cleavage, gel electrophoresis and electron microscopy. At least 90% of the DNA molecules in each sample consisted of sequences that extended from the left end of the viral genome map by variable lengths toward the right end. The average length of DNA is linearly related to the average buoyant density of the incomplete particles from which the DNA is isolated. The results indicate that each capsid contains one DNA molecule. There is also a specific association of the left end of the viral genome with assembled or assembling capsids. The characteristic distributions of Ad7 incomplete particles may result from intracellular pools of assembly intermediates in which the incompletely packaged DNA has been fragmented in vivo or by shear during preparative procedures.     

Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation

The conformational transition of a plasmid DNA, pGEG.GL3 (12.5 kbp, circular), induced by spermine(4+) was studied through the observation of individual DNA by fluorescence microscopy. We deduced the change in the hydrodynamic radius R(H) from an analysis of the Brownian motion of single DNA molecules. R(H) decreases in a continuous manner with an increase in spermine(4+), in contrast to the large discrete on/off change for long linear DNA. Just after the transition to the collapsed state, a small number of DNA molecules tend to form an assembly, which disperses in the bulk solution without precipitation.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

Minichromosome assembly accompanying repair-type DNA synthesis in Xenopus oocytes.

Minichromosomes were assembled by injection of circular DNA into the nucleus of Xenopus oocytes. We observed that, in the course of DNA supercoiling and chromatin assembly, a small percentage of the injected DNA molecules incorporated a radioactive precursor. This DNA synthesis was carried out by aphidicolin-sensitive DNA polymerase, and generated short repair-like patches covalently linked to the injected DNA. We found that the DNA thus repaired was rapidly supercoiled almost to completion within 15 to 30 min after injection, whereas 60 to 120 min were required to supercoil the intact, bulk DNA molecules. Such differential supercoiling kinetics was also observed when UV-damaged DNA was injected. Chromatin assembly, which was characterized by DNA fragment sizes protected from micrococcal nuclease digestion, was consistent with the rapid DNA supercoiling and proceeded more efficiently on the repaired DNA. These results indicate that there are at least two kinetically distinct ways of assembling minichromosomes in the oocyte nucleus, and that the repaired DNA molecules preferentially follow the faster pathway.     

Quantification of Shigella IcsA required for bacterial actin polymerization

Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500-2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella-infected cells.     

Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry

In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.     

c-ABL tyrosine kinase stabilizes RAD51 chromatin association

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.     

Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.     

Preliminary chemical and X-ray studies on the interactions of E. coli DNA with putrescine

The interactions of putrescine, the major diamine in E. coli, with E. coli DNA as a model of phage DNA were studied by melting temperature analysis, equilibrium dialysis and X-ray diffraction with the aid of molecular model building. The chemical analysis of the DNA-putrescine complex shows that the molar binding ratio of putrescine to DNA (phosphate) is nearly 1 to 2. The equilibrium (or reversible) binding of putrescine to DNA was suggested by the fact that the melting temperature increased according to the concentration of added putrescine, and its elevation was not saturated even at the molar ratio of 6 to 1. The equilibrium dialysis experiments indicate that the association constant for the complex is a little smaller than, but in the same order (10(3) liter/mol) as, that of DNA-spermine complex. The binding of putrescine stabilizes the B-form of DNA fiber, which is well preserved even at 66% relative humidity. The distance between the neighboring DNA helices in the wet fiber increased with the increasing degree of hydration, as in the case of native DNA. Unlike spermine, putrescine does not form precipitate upon mixing with DNA in the concentration range for UV measurement, suggesting that the cross-bridge formed by putrescine is intra-double helical. The equilibrium binding of putrescine to DNA, seems to be important for the life cycle of lambda-phage.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.