Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Bosaeus, Niklas; El‐Sagheer, Afaf H.; Brown, Tom; Åkerman, Björn; Nordén, Bengt

doi:10.1093/nar/gku441

Cited by 27 publications

(44 citation statements)

References 40 publications

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“…Laser-tweezers force-spectroscopic data (Bosaeus et al 2012(Bosaeus et al , 2014) on short DNA of variable sequence are here used as basis for discussing the structural and energetic properties of Σ-DNA and to put forward a disproportionation hypothesis.…”

Section: Discussionmentioning

confidence: 99%

“…The Σ-form may be considered a special case of the wider group of stretched DNA forms that have been called 'S-DNA' some of which, though, appear less well defined. In contrast to the Σ-form observed for base-paired GC-rich DNA stretched along the 3′-3′ ends, S-DNA is usually observed as a 70% elongated form when stretching a long plasmid or phage DNA (Cluzel et al 1996;Williams et al 2002) and, at least in AT-rich DNA, S-DNA appears to involve denaturation (Bosaeus et al 2012(Bosaeus et al , 2014. The fact that the Σ-form has so long escaped discovery is thought to be due to that it is first recently stretch experiments on short synthetic DNA have been possible with high accuracy (Bosaeus et al 2012(Bosaeus et al , 2014.…”

Section: Introductionmentioning

confidence: 91%

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A stretched conformation of DNA with a biological role?

Bosaeus

Reymer

Beke‐Somfai

et al. 2017

Quart. Rev. Biophys.

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Abstract. We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair) −1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3′−3′ strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 91%

A stretched conformation of DNA with a biological role?

Bosaeus

Reymer

Beke‐Somfai

et al. 2017

Quart. Rev. Biophys.

Self Cite

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“…Simulations have shown that whereas G-C pairs persist through the transition, A-T pairs often form melted bubbles (30,37). Similarly, various pulling experiments and theoretical models have also demonstrated that at low-to-moderate monovalent salt conditions (<150 mM), AT-rich sequences tend to melt more easily than GC-rich sequences (24,28,29,(65)(66)(67)(68)(69), whereas under high-salt conditions interstrand charge repulsion is reduced, and AT-rich sequences also form S-DNA (31,32). Moreover, cyclic pulling experiments demonstrate that force-induced melting is associated with hysteresis, such that once melted, the complementary strands reanneal slowly (21,28,32,70).…”

Section: High-force Regime: Dap Substitution Decreases the Tension Atmentioning

confidence: 98%

“…The pathway by which DNA becomes overstretched is a function of local stability and is determined by buffer conditions and sequence (26,27). Experiments using relatively short sequences have demonstrated that in physiological salt conditions, AT-rich regions tend to denature upon overstretching, whereas GC-rich regions remain hybridized (28,29). Similarly, molecular dynamic simulations suggest that stronger base pairing in GC-rich regions maintains base registration and minimizes interstrand separation, producing a higher transition force (30).…”

Section: Introductionmentioning

confidence: 99%

Nanomechanics of Diaminopurine-Substituted DNA

Cristofalo

Kovari

Corti

et al. 2019

Biophysical Journal

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2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

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“…36 At the low-salt conditions used in these experiments, this transition occurs at about 62 pN and represents a conversion of DNA from dsDNA to ssDNA as the DNA is destabilized by force and primarily peels from its free end. [37][38][39][40][41][42] The force-extension profiles of ssDNA and dsDNA cross at $6 pN [ Fig. 1(B)].…”

Section: Pol III Core Activity At Constant Forcementioning

confidence: 99%

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Cited by 27 publications

References 40 publications

A stretched conformation of DNA with a biological role?

A stretched conformation of DNA with a biological role?

Nanomechanics of Diaminopurine-Substituted DNA

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

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