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. 2012 Sep 18;109(38):15179-84.
doi: 10.1073/pnas.1213172109. Epub 2012 Sep 4.

Tension induces a base-paired overstretched DNA conformation

Niklas Bosaeus  1 , Afaf H El-Sagheer, Tom Brown, Steven B Smith, Björn Akerman, Carlos Bustamante, Bengt Nordén
Affiliations

Tension induces a base-paired overstretched DNA conformation

Niklas Bosaeus et al. Proc Natl Acad Sci U S A. .

Abstract

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106:18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schemes of DNA constructs, covalent linkage and DNA-bead assembly. (A) DNA constructs consisting of a central double helical part (kernel) and two single-stranded extensions (handles). (a) Kernel strands are complementary 60 nucleotides long (for sequences, see Fig. S1), forming the GC-rich OligoGC1 and OligoGC2 and the AT-rich OligoAT. Each strand is extended at the 3′ end by terminal transferase into a single-strand handle containing biotinylated uracil bases (triangles) or digoxigenin (circles). Clickable (64 bp) kernels contain at the end of one strand an alkyne group (≡) and, on the complementary strand, a matching azide group (N3) prepared for click linking (see Materials and Methods). (b) Kernel covalently sealed at 5′ end of biotinylated strand (e.g., in 5′OligoGC1, click-link marked by small black dot). (c) Kernel sealed at 3′ end (e.g., in 3′OligoGC1). (d) Kernel sealed at both ends (e.g., 3′5′OligoGC1). For synthesis, see SI Text. (B) Bead attachment of DNA constructs and hypothetical overstretched states of kernel. (i) Partially molten kernel with base pairs remaining at one end as one strand peels off at opposite end (peeling model), (ii) Partially molten kernel interior, with base pairs remaining at both ends (internal melting model), (iii) Overstretched base paired duplex. (iv) Fully denatured kernel, with remaining covalent linker.
Fig. 2.
Fig. 2.
Mechanical stretching of single nonclicked 60 base-pair OligoGC1 molecule (sequence in Fig. S1) in 1 M NaCl. (A) Force-distance plot during one cycle of pulling (blue) and relaxation (red). The DNA duplex exhibits a bistability between 61 and 65 pN, with no detectable hysteresis between the pulling and relaxation parts of the cycle. Inset: bistability region in detail. The duplex switches between the overstretched form (low force) and the shorter native B-form (high force) during both pulling and relaxation. (B) Force-distance plot during a single pulling, illustrating method of data analysis. Each data point is assigned to the B-form or the overstretched state depending on its proximity to the lines fitted to the force-distance data at low (green) and high force (brown), respectively. (C) Probability P of finding the molecule in the overstretched state as a function of force. Experimental data (circles) pooled from 16 pull-and-relax cycles from the same OligoGC1 molecule. The curve is a fit to a two-state model P = 1/(1 + eb-Fa) (42) and the transition force Ftr corresponds to P = 0.5 (giving Ftr = 63.75 pN for this particular OligoGC1 molecule). (D) Conversion dynamics between the overstretched and native forms. Time trace of force in the overstretching region for OligoGC1 molecule measured at 1 kHz with trap held stationary. Low force corresponds to overstretched form and high force to shorter B-form. The histogram (to the right) gives the distribution of data points during studied time interval (1 s). The bimodal Gaussian distribution supports the interpretation that the transition is two-state in nature.
Fig. 3.
Fig. 3.
Force-distance data for GC-rich and AT-rich duplexes covalently sealed at one end (1 M NaCl). Main panel shows one cycle of pulling (blue) and relaxation (red) of single DNA molecule. Inset shows the details of the force-induced transitions for the two different sequences. (A) 5′-clicked OligoGC1. During pulling reversible switching occurs between two states at about 60 pN. At 74 pN an abrupt decrease in force (denoted strand dissociation) occurs during relaxation. Inset i shows switching region in detail, and inset ii that the transition at 74 pN occurs in a single step. (B) 5′-clicked OligoAT. During pulling there is an abrupt transition to an extended state at 62 pN, and hysteresis during relaxation consistent with melting. Rehybridization to B-form is observed as a return to the pulling curve at about 20 pN. Inset i shows the 62 pN transition in detail. Before the abrupt extension, at force Fm, reversible switching to a longer intermediate state is observed. Inset ii shows that the rehybridization at 20 pN occurs in a single step.
Fig. 4.
Fig. 4.
Probing base-pairing characteristics of overstretched state with glyoxal-reactivity. (A) 5′-clicked AT-rich oligonucleotide, 5′OligoAT. Pull-relax cycles in presence of 0.5 M glyoxal before and after reaction. Blue curve is the first pull cycle before DNA reacts with glyoxal (t0): the unmodified molecule melts at about 60 pN (inset i). After the DNA molecule is melted the force is lowered to approximately 40 pN where it does not reanneal and is held there for some amount of time to allow exposure of the single strands to glyoxal. After this exposure time, the force is lowered to 20 pN, where the molecule rehybridizes within a few seconds. Green curve (t50 s) shows the same molecule after it has been exposed in the melted state to glyoxal a total of 50 s. The molecule is seen to melt at about 49 pN (inset ii), showing that the glyoxal-induced base reaction lowers the melting force of the molecule. The red curve shows the molecule after it is exposed in its melted state to glyoxal for 2.5 min. During pull DNA first melts partially (note absence of bistability) before it melts completely at 41 pN (inset iii). (B) Double-clicked GC-rich oligonucleotide, 3′5′OligoGC1. Blue curve shows first pull in presence of glyoxal; green curve shows pull of the same molecule after 5.5 min of exposure to glyoxal while in the overstretched state (> 70 pN). Curves have been horizontally compensated for instrument drift (< 4 nm/ min) to simplify comparison between different pull cycles.
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