Search

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Central biological processes involve continuous mechanical manipulation of DNA. In cells, DNA is constantly twisted, bended and stretched by numerous proteins mediating genome compaction, gene regulation, expression, and DNA repair. Consequently, to understand these processes, it is imperative to have an in-depth understanding of how DNA complies to mechanical stress. The helical structure and the sequence of DNA, two physical features that have a strong impact on protein-DNA interactions, are not incorporated in the current quantitative descriptions of DNA elasticity. Here we connect well-defined force-extension measurements with a novel structure-guided model for DNA elasticity, the twistable worm like chain model. This analytical description incorporates the essential physical characteristics of DNA, including how its helicity depends on extension. In addition, at forces exceeding ∼65 pN, when DNA overstretches and melts, our experimental assay exposes rich features that can be fully attributed to the underlying base sequence. An equilibrium thermodynamic model is presented that quantitatively captures this melting behaviour solely based on the knowledge of DNA sequence and elasticity. These results offer a new standard description for the mechanics of DNA and enable deeper quantitative insight into the physical interactions of DNA-associated proteins.

DNA replication needs to be both faithful and processive. DNA polymerases replicate DNA, using a template strand to add the complementary nucleotide to the synthesizing primer (polymerization). Family A DNA polymerases, like T7 DNA polymerase (DNAp), boost their fidelity by proofreading the primer. In the event of a mismatch, the erroneous nucleotide is excised at the exonuclease active site (proofreading). Proper balance between polymerization and proofreading is essential to achieve faithful but productive replication.Here we report on a single-molecule study of DNAp using optical tweezers. In our setup we can directly measure the individual rates of replicating, proofreading and pausing single DNA polymerases at various tensions. With a spatial and temporal resolution of 25 bp and 0.2 sec, we can study DNAp in far more detail than previous single-molecule studies.We find that the rate of polymerization decreases with tension on the DNA but, notably, doesn’t stall at high tension. Next, the rate of processive proofreading is constant over all measured tensions which, surprisingly, include tensions of only a few pN. This means that the proofreading state of DNAp is highly accessible, independent of perturbations of the primer-template structure, and that during proofreading tens of nucleotides at a time are removed.Our observations indicate that the current kinetic model for DNAp is not sufficient. The length of the pauses in between events are distributed as a double exponential, arguing against a single inactive state. Therefore, we introduce an updated model that contains an extra state that might play a role in balancing polymerization and proofreading. The conclusion seems that the cost of being right (i.e. error-free replication) outweighs the price a cell pays (i.e. the regular removal of correct base pairs) for picking up errors during replication.

DNA undergoes a structural transition at a tension of 65 pN, were the polymer gains 70% of its contour length. The molecular basis of this overstretching transition has been elucidated using a combination of fluorescence microscopy and optical tweezers: At a tension of 65 pN, the DNA undergoes a nucleation-limited force-induced melting transition, in which the DNA strands gradually fray from the DNA's extremities during progression of overstretching [1].Here we demonstrate that inhibition of this fraying process from one of the two DNA ends leads to a single deterministic melting front, which allows us to correlate the force signal in the overstretching plateau to the melted sequence.We show that the propagation of the melting front progresses in bursts involving cooperative unbinding of multiple base-pairs. We furthermore proof that this burst-wise melting is an equilibrium process that is completely determined by the DNA sequence. Applying an equilibrium molecular stick-slip theory, we obtain a good agreement in both the force at which the DNA molecule starts to denature and the location of the individual melting bursts. We demonstrate that this theory, with the underlying DNA sequence as input, is able to predict the force-induced melting behavior.Furthermore, we explore individual melting bursts by monitoring the force level of a DNA close to a melting event, and observe a bistability between two levels of the melting process. The time scale between the melting-reannealing events provides us with insight into the underlying local energy landscape close to a melting burst.[1] van Mameren et al., Unraveling the structure of DNA during overstretching using multicolor, single-molecule fluorescence imaging, PNAS (in press) 2009

Single-molecule manipulation techniques have provided a comprehensive understanding of the elastic behavior of DNA. We now know that its double-helical structure yields a significant persistence against bending on length scales of 150 base pairs and smaller. Interestingly, optical tweezers studies have also revealed, that at 65pN DNA undergoes a phase transition to another structure with significantly different elastic properties. Over a very narrow force range the polymer gains ∼70% in contour length, and becomes significantly more flexible.Until now the basic microscopic structure of overstretched DNA is under debate. Two qualitatively different models disagree on the molecular mechanism of the overstretching transition. The first one suggests that the DNA double helix unwinds to form a new structure, named S-DNA, which is usually depicted as a ladder with intact base pairing. The second model states that DNA overstretching is a force-induced melting transition, in which the hydrogen bonds between the two strands gradually break to yield single-stranded DNA, similar to thermal melting.Using a combination of fluorescence microscopy and optical tweezers we directly visualize the DNA overstretching transition and demonstrate that it is driven by melting of the double-stranded DNA. In the experiments we use intercalating dyes and fluorescently labeled single-stranded binding proteins to specifically visualize double- and single-stranded segments in DNA molecules undergoing the transition. Our data unambiguously show that the overstretching transition comprises a gradual conversion from double-stranded to single-stranded DNA, in agreement with the force-induced melting model. Interestingly, not predicted by either model, we found that melting is nucleation-limited, typically initiating from DNA extremities and nicks.

DNA-proteins interactions can be studied in great detail when precise and dynamic control of the mechanical state of a single DNA molecule can be achieved together with direct visualization of proteins interacting with the DNA. Our custom-built experimental setup combines optical trapping and wide-field epifluorescence microscopy. In this setup we can visualize single DNA-bound proteins (see figure) with an accuracy of tens of nanometers while at the same time we can control the tension applied on the DNA at a sub-PicoNewton level.Here we report the limit of localization accuracy in combined DNA-trapping/fluorescence experiments by fitting a point-spread function to the fluorescence image of single DNA-bound proteins.We investigate the impact of DNA dynamics on the maximum attainable accuracy for the localization of these DNA-bound fluorescent proteins (in this case a Ca2+ inactivated restriction enzyme EcoRV). In particular, we study the effect of tension on the DNA and identify the force regime in which single-proteins can be localized with super-resolution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

DNA replication is an essential cell process in which the genetic information is copied by replicative DNA polymerases (DNAp). The molecular basis of DNA replication is the addition of nucleotides by DNAp to a growing primer, using single-stranded DNA as a template. High fidelity of the processive T7 DNA polymerase comes from nucleotide selection at the polymerase active site, but is increased several orders of magnitude by an additional intrinsic proofreading ability. In this kinetic process, a partly melted primer shuttles to the exonuclease active site where incorporated mismatches are excised. After excision of erroneous nucleotides, the trimmed primer can shuttle back to the polymerase active site to resume replication. Elucidating the mechanism of the shuttling between these two activities of DNAp is essential for understanding the proofreading mechanism of DNA polymerases.Transfer of the primer to the exonuclease active site is induced by disruption of the primer-template structure upon the incorporation of a mismatch. Application of tension to the DNA also destabilizes the primer-template structure and can therefore be used to shift the fine-tuned balance between polymerization and proofreading (Wuite et al, 2001; Ibarra et al, 2009).Using optical tweezers, we study the kinetic coordination between exonuclease and polymerase activities, while applying different tensions. In these experiments we observe an additional waiting state between proofreading activities, during which the DNAp remains bound to the DNA. The force-dependent rate out of this state suggests that DNAp enters a state comparable to RNA polymerase backtracked state, which was shown to play a role in tuning the fidelity (Shaevitz et al, 2003). We speculate that our observed waiting state might play a similar role in the fidelity of DNA polymerase.