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1.
Taylor ER McGowan CH 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(10):3757-3762
Recombination-mediated repair plays a central role in maintaining genomic integrity during DNA replication. The human Mus81-Eme1 endonuclease is involved in recombination repair, but the exact structures it acts on in vivo are not known. Using kinetic and enzymatic analysis of highly purified recombinant enzyme, we find that Mus81-Eme1 catalyzes coordinate bilateral cleavage of model Holliday-junction structures. Using a self-limiting, cruciform-containing substrate, we demonstrate that bilateral cleavage occurs sequentially within the lifetime of the enzyme-substrate complex. Coordinate bilateral cleavage is promoted by the highly cooperative nature of the enzyme and results in symmetrical cleavage of a cruciform structure, thus, Mus81-Eme1 can ensure coordinate, bilateral cleavage of Holliday junction-like structures. 相似文献
2.
Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases
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Charvin G Bensimon D Croquette V 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(17):9820-9825
Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of approximately 2.9 s-1, Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of approximately 2.4 s-1. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid. 相似文献
3.
Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions
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Kuzmenkina EV Heyes CD Nienhaus GU 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(43):15471-15476
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through F?rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximately equal to 20 micros at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximately equal to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants. 相似文献
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5.
Stroh C Wang H Bash R Ashcroft B Nelson J Gruber H Lohr D Lindsay SM Hinterdorfer P 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(34):12503-12507
Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody-antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process. 相似文献
6.
Singh-Zocchi M Dixit S Ivanov V Zocchi G 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(13):7605-7610
We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe. 相似文献
7.
Single-molecule mechanics of mussel adhesion 总被引:3,自引:0,他引:3
Lee H Scherer NF Messersmith PB 《Proceedings of the National Academy of Sciences of the United States of America》2006,103(35):12999-13003
The glue proteins secreted by marine mussels bind strongly to virtually all inorganic and organic surfaces in aqueous environments in which most adhesives function poorly. Studies of these functionally unique proteins have revealed the presence of the unusual amino acid 3,4-dihydroxy-L-phenylalanine (dopa), which is formed by posttranslational modification of tyrosine. However, the detailed binding mechanisms of dopa remain unknown, and the chemical basis for mussels' ability to adhere to both inorganic and organic surfaces has never been fully explained. Herein, we report a single-molecule study of the substrate and oxidation-dependent adhesive properties of dopa. Atomic force microscopy (AFM) measurements of a single dopa residue contacting a wet metal oxide surface reveal a surprisingly high strength yet fully reversible, noncovalent interaction. The magnitude of the bond dissociation energy as well as the inability to observe this interaction with tyrosine suggests that dopa is critical to adhesion and that the binding mechanism is not hydrogen bond formation. Oxidation of dopa, as occurs during curing of the secreted mussel glue, dramatically reduces the strength of the interaction to metal oxide but results in high strength irreversible covalent bond formation to an organic surface. A new picture of the interfacial adhesive role of dopa emerges from these studies, in which dopa exploits a remarkable combination of high strength and chemical multifunctionality to accomplish adhesion to substrates of widely varying composition from organic to metallic. 相似文献
8.
Bokinsky G Rueda D Misra VK Rhodes MM Gordus A Babcock HP Walter NG Zhuang X 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(16):9302-9307
How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions. 相似文献
9.
10.
Single-molecule observation of DNA charge transfer 总被引:1,自引:0,他引:1
Takada T Fujitsuka M Majima T 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(27):11179-11183
DNA charge transfer highly depends on the electronic interaction between base pairs and reflects the difference in the base composition and sequence. For the purpose of investigating the charge transfer process of individual DNA molecules and the optical readout of DNA information at the single-molecule level, we performed single-molecule observation of the DNA charge transfer process by using single-molecule fluorescence spectroscopy. The DNA charge transfer process, leading to the oxidation of the fluorescent dye, was explored by monitoring the on-off signal of the dye after the charge injection by the excitation of a photosensitizer. The photobleaching efficiency of the dyes by the DNA charge transfer specifically depended on the base sequence and mismatch base pair, demonstrating the discrimination of the individual DNA information. Based on this approach, the optical readout of a single-base mismatch contained in a target DNA was performed at the single-molecule level. 相似文献
11.
Gordon MP Ha T Selvin PR 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(17):6462-6465
Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a “gap” in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10–20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms. 相似文献
12.
Mark A. Hilton Harris W. Manning Ireneusz Grniak Sonia K. Brady Madeline M. Johnson Jochen Zimmer Matthew J. Lang 《Proceedings of the National Academy of Sciences of the United States of America》2022,119(40)
Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.Cellulose is an integral structural component utilized by several kingdoms of life for its high mechanical strength and chemical stability (1, 2). Lately, cellulose’s contribution to cell walls and microbial mats has garnered great interest as cellulosic biofuels become increasingly competitive (3) and as cellulose-stabilized bacterial biofilms are shown to play significant roles in pathogenesis (4–6). Cellulose is a polysaccharide composed of repeating glucosyl units linked by β (1–4) glycosidic bonds. Investigations of its crystalline fibrillar form show that strands are linearly arranged and flat (7). In gram-negative bacteria, cellulose is manufactured through a multisubunit transenvelope bacterial cellulose synthase (Bcs) complex containing the evolutionarily conserved (8) catalytic BcsA subunit and an inner membrane–anchored domain known as BcsB (9). The membrane-embedded BcsAB complex likely interacts with BcsC in the outer membrane to form a continuous transmembrane conduit for cellulose secretion. In vitro functional and structural studies on the purified Rhodobacter sphaeroides BcsAB complex revealed that it alone is sufficient for cellulose synthesis and secretion across the inner bacterial membrane (9). BcsA is allosterically activated by cyclic diguanosine monophosphate (c-d-GMP), enabling its glycosyltransferase domain to bind the Mg2+-coordinated uridine diphosphate glucose (UDP-glc) substrate (10, 11). UDP-glc reacts with and elongates the nonreducing terminal end of the cellulose chain one glucose unit at a time, releasing UDP by-product afterward (12). Subsequently, the polymer translocates through a transmembrane channel formed by BcsA and is likely guided into the periplasmic space by BcsB (13). Surprisingly, the degree of processive polymerization from cellulose synthases of different origins ranges from hundreds to thousands of glucose units (14, 15).The cellulose polymer produced by BcsAB is a main component of biofilm matrices that encase sessile bacterial colonies, particularly among enterobacteria (6). Adherent bacterial populations besiege industrial systems by plugging filters, corroding metal surfaces, and fouling pipes (16). In healthcare settings, robust biofilms are responsible for ∼65% of nosocomial infections and are considerably resistant to antimicrobial treatments (5, 17). Inhibiting the production of extracellular polymeric substances, such as polysaccharides, is a strong potential antibiofilm strategy (18). Thus, a molecular understanding of bacterial cellulose synthesis is paramount for the development of powerful antibacterial agents.BcsAB has been well described by crystallographic snapshots and in vitro analyses; however, these methods lack details of biosynthesis at the molecular level (13, 19). Extensive work has been done to characterize cellulose synthesis and the properties of cellulose (1, 2, 9, 10, 13, 19–21). Cellulose, as an abundant wall polymer of vascular plants, has been described substantially in its amorphous and crystalline forms using X-ray diffraction (22), molecular dynamics simulations (22, 23), and atomic force microscopy (24), among other methods (20, 25, 26). In all cases, studies included cellulose aggregates or atomistic models. While it is known that BcsAB produces high-molecular-weight amorphous cellulose (8), the physical and dynamic properties of single cellulose chain synthesis leading to this structure have not been characterized.A real-time, molecular-scale analysis of cellulose synthesis and single-chain cellulose offers essential insight into the formation and structural qualities of this abundant biopolymer. Biosynthesis requires multiple elements, including activated glucose, c-d-GMP, and Mg2+. Furthermore, product transport and product microstructure may also impact biosynthesis. Cellulose production may be impacted by mechanical force, as seen in other molecular machines (27–29). Here, we use optical tweezers to directly probe mechanical and catalytic activity of single BcsAB molecules and their single-strand cellulose polymer products. 相似文献
13.
Steiner M Karunatilaka KS Sigel RK Rueda D 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(37):13853-13858
Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a Mg2+-dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM Mg2+ and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing Mg2+ concentrations, enabling the enzyme to reach its active state. 相似文献
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15.
Ruth Kellner Hagen Hofmann Alessandro Barducci Bengt Wunderlich Daniel Nettels Benjamin Schuler 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(37):13355-13360
Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ–DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Maintaining protein homeostasis in vivo requires a tight regulation of protein folding to prevent misfolding and aggregation. Molecular chaperones have evolved as an essential part of the cellular machinery that facilitates such processes in the complex and crowded environment of a living cell (1, 2). To assist protein folding, many chaperones proceed through complex conformational cycles in an ATP-dependent manner (3–5). For several chaperone systems, these cycles have been investigated in great detail by experiment and simulation (6–8). A remarkable example are the heat shock protein (Hsp) 70 chaperones, which are essential in prokaryotes and eukaryotes and are involved in co-translational folding, refolding of misfolded and aggregated proteins, protein translocation, and protein degradation (9). The Hsp70 chaperone DnaK from Escherichia coli together with its co-chaperone DnaJ and the nucleotide exchange factor GrpE form an ATP-driven catalytic reaction cycle (7) (Fig. 1A). Many denatured or misfolded substrate proteins are first captured by DnaJ and subsequently transferred to the DnaK–ATP complex, with DnaK in an open conformation. Substrate and DnaJ synergistically trigger DnaK’s ATPase activity, which leads to locking of the substrate in the DnaK–ADP complex, with DnaK in the closed conformation. Driven by the following GrpE-catalyzed ADP–ATP exchange, the DnaK–substrate complex dissociates (10). Since this ATP-driven cycle can even solubilize protein aggregates (11, 12), substantial forces must be transduced to the substrate protein (13–15). However, as for other chaperone systems (16), surprisingly little is known about how these forces and the resulting constraints of the underlying free energy surfaces affect the conformations of the denatured or misfolded substrate proteins. To better understand this important link between chaperone action and function, we probed the conformation of a substrate protein along the different stages of the chaperone cycle of DnaK with single-molecule Förster resonance energy transfer (smFRET), correlation spectroscopy, and microfluidic mixing.Open in a separate windowFig. 1.DnaK expands the denatured substrate protein. (A) Illustration of the DnaK–ATPase cycle. (B) Surface representation of rhodanese (PDB ID code 1RHS) with the subdomains indicated in different gray levels and the label positions of fluorescent dyes for single-molecule FRET measurements shown schematically. (C) FRET efficiency histograms of native rhodanese (gray) and denatured rhodanese under native conditions transiently populated in the microfluidic mixer (colored, measured 125 ms after dilution of rhodanese into native conditions). (D) FRET efficiency histograms of DnaJ–rhodanese complexes (0.5 µM DnaJ). (E) FRET efficiency histograms of DnaK–rhodanese complexes (0.5 µM DnaJ, 10 µM DnaK, and 1 mM ATP; DnaK and DnaJ were added simultaneously to rhodanese). Black lines indicate the DnaK–rhodanese complex population resulting from a fit that takes into account the residual population of refolded and DnaJ-bound rhodanese. The vertical lines in C–E indicate the positions of the FRET efficiency peaks of the native population of the respective rhodanese variants. The small populations at zero transfer efficiency in D (note the axis scaling and the small amplitudes of this population compared with E) originate from incomplete elimination of molecules with inactive acceptor fluorophores by pulsed interleaved excitation. 相似文献
16.
Walter Reisner Niels B. Larsen Asli Silahtaroglu Anders Kristensen Niels Tommerup Jonas O. Tegenfeldt Henrik Flyvbjerg 《Proceedings of the National Academy of Sciences of the United States of America》2010,107(30):13294-13299
Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent “barcode” corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells. 相似文献
17.
Wang YM Tegenfeldt JO Reisner W Riehn R Guan XJ Guo L Golding I Cox EC Sturm J Austin RH 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(28):9796-9801
We have performed single-molecule studies of GFP-LacI repressor proteins bound to bacteriophage lambda DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths. 相似文献
18.
Hagen Hofmann Frank Hillger Shawn H. Pfeil Armin Hoffmann Daniel Streich Dominik Haenni Daniel Nettels Everett A. Lipman Benjamin Schuler 《Proceedings of the National Academy of Sciences of the United States of America》2010,107(26):11793-11798
Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra- and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage. 相似文献
19.
Single-molecule tracking of mRNA exiting from RNA polymerase II 总被引:3,自引:1,他引:2
Andrecka J Lewis R Brückner F Lehmann E Cramer P Michaelis J 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(1):135-140
Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5' end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5'-capping enzyme and other RNA processing factors. 相似文献
20.
Anna K. Wo
niak Gunnar F. Schrder Helmut Grubmüller Claus A. M. Seidel Filipp Oesterhelt 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(47):18337-18342
We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye–dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16°. For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32° ± 6°, 56° ± 4° and 73 ± 2°, respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail. 相似文献