Structural Transitions of a Twisted and Stretched DNA Molecule

Single-molecule manipulation studies have revealed that doublestranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates Ϸ1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double-and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to singlestranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.DNA melting ͉ fluorescence microscopy ͉ optical trapping ͉ single-molecule techniques ͉ single-stranded DNA

show abstract

“…1A) (2,3). A similar transition is observed when the DNA is torsionally constrained (4,5), then occurring at a force of around 110 pN (Fig. 1B).…”

mentioning

confidence: 50%

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

Mameren

Groß

Farge

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

266

294

View full text Add to dashboard Cite

show abstract

“…The present study focuses on another form of nucleic acid duplexes, recently revealed in single-molecule experiments that twisted and stretched double-stranded DNA with magnetic or optical beads attached to the ends (14,25,26). In the overtwisting case, these experiments produced a structure that was hypothesized to be akin to, (and thereby to somewhat vindicate), an early model of DNA proposed by Pauling (27) (P-DNA).…”

mentioning

confidence: 99%

On structural transitions, thermodynamic equilibrium, and the phase diagram of DNA and RNA duplexes under torque and tension

Wereszczynski

Andricioaei

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A precise understanding of the flexibility of double stranded nucleic acids and the nature of their deformed conformations induced by external forces is important for a wide range of biological processes including transcriptional regulation, supercoil and catenane removal, and site-specific recombination. We present, at atomic resolution, a simulation of the dynamics involved in the transitions from B-DNA and A-RNA to Pauling (P) forms and to denatured states driven by application of external torque and tension. We then calculate the free energy profile along a B-to P-transition coordinate and from it, compute a reversible pathway, i.e., an isotherm of tension and torque pairs required to maintain P-DNA in equilibrium. The reversible isotherm maps correctly onto a phase diagram derived from single molecule experiments, and yields values of elongation, twist, and twist-stretch coupling in agreement with measured values. We also show that configurational entropy compensates significantly for the large electrostatic energy increase due to closer-packed P backbones. A similar set of simulations applied to RNA are used to predict a novel structure, P-RNA, with its associated free energy, equilibrium tension, torque and structural parameters, and to assign the location, on the phase-diagram,

show abstract

“…Overstretching is of crucial importance for the biological function of DNA: the bacterial protein RecA elongates DNA by a factor of 1.5 upon binding [3,4,5], a mechanism central to homologous recombination and to chromosomal segregation during cell division [6]. However, the nature of the overstretched state remains a source of considerable controversy: some think overstretched DNA in vitro to be a hybridized form called S-DNA (the 'Bto-S' picture) [7,8,9,10,11,12], while a competing picture considers overstretching to signal a conversion to unhybrizided single strands (the 'force-melting' picture) [13,14,15,16,17,18]. We therefore lack full understanding of the basic mechanical and thermodynamic properties of a molecule of central importance to biology and of rapidly increasing importance to technology [19,20,21,22].…”

Section: Introductionmentioning

confidence: 99%

Stretching chimeric DNA: A test for the putative S-form

Whitelam

Pronk

Geissler

2008

The Journal of Chemical Physics

View full text Add to dashboard Cite

Double-stranded DNA 'overstretches' at a pulling force of about 65 pN, increasing in length by a factor of 1.7. The nature of the overstretched state is unknown, despite its considerable importance for DNA's biological function and technological application. Overstretching is thought by some to be a force-induced denaturation, and by others to consist of a transition to an elongated, hybridized state called S-DNA. Within a statistical mechanical model we consider the effect upon overstretching of extreme sequence heterogeneity. 'Chimeric' sequences possessing halves of markedly different AT composition elongate under fixed external conditions via distinct, spatially segregated transitions. The corresponding force-extension data display two plateaux at forces whose difference varies with pulling rate in a manner that depends qualitatively upon whether the hybridized S-form is accessible. This observation implies a test for S-DNA that could be performed in experiment. Our results suggest that qualitatively different, spatially segregated conformational transitions can occur at a single thermodynamic state within single molecules of DNA.

show abstract

Structural Transitions of a Twisted and Stretched DNA Molecule

Cited by 265 publications

References 9 publications

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

On structural transitions, thermodynamic equilibrium, and the phase diagram of DNA and RNA duplexes under torque and tension

Stretching chimeric DNA: A test for the putative S-form

Contact Info

Product

Resources

About