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   

Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins

The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single‐molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high‐order molecular structures. For example, single‐stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two‐state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm‐like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two‐state transition between mechanically folded and unfolded states (such as proteins). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1193–1199, 2014.     

An integrative approach for the optical sequencing of single DNA molecules

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.     

The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series of conformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across the entire DNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found that fingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (～5.3 s^?1), and comparable to the rate of fingers-opening after polymerisation (3.4 s^?1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3′ end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 → 29 s^?1), demonstrating that the 3′-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.     

Thermodynamics of single strand DNA base stacking

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<-->U transition in this temperature range. The transition properties of the ssDNA sequences with 相似文献   

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.     

Kinetic regulation of single DNA molecule denaturation by T4 gene 32 protein structural domains

Bacteriophage T4 gene 32 protein (gp32) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), gp32 does not. Using single molecule force spectroscopy, we show for the first time that gp32 is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of gp32 and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate gp32's helix-destabilizing capabilities. Whereas the full-length protein exhibits very slow denaturation kinetics, a truncate lacking the acidic C-domain exhibits much faster kinetics. This may reflect a steric blockage of the DNA binding site and/or a conformational change associated with this domain. Additional removal of the N-domain, which is needed for binding cooperativity, further increases the DNA denaturation rate, suggesting that both of these domains are critical to the regulation of gp32's helix-destabilization capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the helix-coil transition free energy in the presence of these proteins for the first time.     

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.     

Mechanical stability of multidomain proteins and novel mechanical clamps

We estimate the size of mechanostability for 318 multidomain proteins which are single-chain and contain up to 1021 amino acids. We predict existence of novel types of mechanical clamps in which interdomain contacts play an essential role. Mechanical clamps are structural regions which are the primary source of a protein's resistance to pulling. Among these clamps there is one that opposes tensile stress due to two domains swinging apart. This movement strains and then ruptures the contacts that hold the two domains together. Another clamp also involves tensile stress but it originates from an immobilization of a structural region by a surrounding knot-loop (without involving any disulfide bonds). Still another mechanism involves shear between helical regions belonging to two domains. We also consider the amyloid-prone cystatin C which provides an example of a two-chain 3D domain-swapped protein. We predict that this protein should withstand remarkably large stress, perhaps of order 800 pN, when inducing a shearing strain. The survey is generated through molecular dynamics simulations performed within a structure-based coarse grained model.     

Thermophilic topoisomerase I on a single DNA molecule

Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix. An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases. We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles. We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA. The relaxation rate depended sensitively on the applied force and the protein concentration. These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids. Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T.maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA. Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase.     

The double-stranded DNA stability in presence of a flexible polymer

The mixture of the short segments of double-stranded DNA and a flexible polymer are addressed. It is shown that in the condensed phase, rigid DNA molecules exhibit transition between isotropic and orientationally ordered phases. It is shown that orientational ordering stabilizes the secondary structure of double-stranded DNA that could be relevant for the regulation of the gene expression at the condensed state of DNA.     

A single molecule assay for measuring site-specific DNA cleavage

Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.     

Purification of total cellular DNA from a single plant

A procedure is outlined for purifying DNA from a single plant. A crude organelle pellet consisting of nuclei, chromatin, chloroplasts, and mitochondria is prepared, suspended, and immediately lysed with detergents. The DNA is separated from RNA, protein, and polysaccharides by banding it in CsCl density equilibrium gradients. Ethidium bromide is included in all buffers to act as an inhibitor of DNAase activity. The DNA prepared in this manner can be digested with restriction endonucleases, separated by gel electrophoresis, and used to identify specific genes by hybridization of cloned DNA sequences.These experiments were supported by Grant DEB79-2298 from the National Science Foundation and Grant 59-2133-0-1-489-0 from the USDA Competitive Research Grants Program.     

A cooperative transition from the semi-flexible to the flexible regime of polymer elasticity: Mitoxantrone-induced DNA condensation

We report a high cooperative transition from the semi-flexible to the flexible regime of polymer elasticity during the interaction of the DNA molecule with the chemotherapeutic drug Mitoxantrone (MTX). By using single molecule force spectroscopy, we show that the force-extension curves of the DNA-MTX complexes deviate from the typical worm-like chain behavior as the MTX concentration in the sample increases, becoming straight lines for sufficiently high drug concentrations. The behavior of the radius of gyration of the complexes as a function of the bound MTX concentration was used to quantitatively investigate the cooperativity of the condensation process. The present methodology can be promptly applied to other ligands that condense the DNA molecule upon binding, opening new possibilities in the investigation of this type of process and, more generally, in the investigation of phase transitions in polymer physics.     

Bead size effects on protein-mediated DNA looping in tethered-particle motion experiments

Tethered particle motion (TPM) has become an important tool for single-molecule studies of biomolecules; however, concerns remain that the method may alter the dynamics of the biophysical process under study. We investigate the effect of the attached microsphere on an illustrative biological example: the formation and breakdown of protein-mediated DNA loops in the lac repressor system. By comparing data from a conventional TPM experiment with 800 nm polystyrene beads and dark-field TPM using 50 nm Au nanoparticles, we found that the lifetimes of the looped and unlooped states are only weakly modified, less than two-fold, by the presence of the large bead. This is consistent with our expectation of weak excluded-volume effects and hydrodynamic surface interactions from the cover glass and microsphere.     

Thermal stability of DNA with interstrand crosslinks

Although many anticancer drugs exert their biological activity by forming DNA interstrand crosslinks (ICLs), the thermodynamics of biologically relevant long crosslinked DNAs has not been intensively studied in contrast to short duplexes. Here, we carry out computer modeling of the shift of melting temperature of long DNAs caused by ICLs taking into account crosslinking effect in itself and concomitant local alterations in the free energy (δG) of the helix-coil transition at sites of ICLs. Depending on δG, DNA interstrand crosslinks at per nucleotide concentration r = 0.05 can change the melting temperature by value from -17 to +47°C, and the influence weakly depends on DNA sequence and GC content. A change in melting temperature caused by introduction of interstrand crosslinking in modified DNA at sites of modifications also depends on δG and varies from 0 to +12°C. Comparison with experiment for the three platinum crosslinking compounds demonstrates utility of the theoretical method for understanding how crosslinking compounds can influence the melting behavior. On the basis of the method, interdependence of local distortions and crosslinking in itself was studied for thermal effect of ICLs. A method for evaluating the nature of the structural alteration that produces a change in thermal stability for short crosslinked DNA is also proposed. The methods can be used for comparative thermodynamic characterization of various DNA crosslinking agents.