Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements

Abstract

Double-stranded DNA is a dynamic molecule whose structure can change depending on conditions. While there is consensus in the literature about many structures DNA can have, the state of highlystretched DNA is still not clear. Several groups have shown that DNA in the torsion-unconstrained B-form undergoes an "overstretching" transition at a stretching force of around 65 pN, which leads to approximately 1.7-fold elongation of the DNA contour length. Recent experiments have revealed that two distinct structural transitions are involved in the overstretching process: (i) a hysteretic "peeling" off one strand from its complementary strand, and (ii) a nonhysteretic transition that leads to an undetermined DNA structure. We report the first simultaneous determination of the entropy (ΔS) and enthalpy changes (ΔH) pertaining to these respective transitions. For the hysteretic peeling transition, we determined ΔS ∼ 20 cal∕ðK:molÞ and ΔH ∼ 7 kcal∕mol. In the case of the nonhysteretic transition, ΔS ∼ −3 cal∕ðK:molÞ and ΔH ∼ 1 kcal∕mol. Furthermore, the response of the transition force to salt concentration implies that the two DNA strands are spatially separated after the hysteretic peeling transition. In contrast, the corresponding response after the nonhysteretic transition indicated that the strands remained in close proximity. The selection between the two transitions depends on DNA base-pair stability, and it can be illustrated by a multidimensional phase diagram. Our results provide important insights into the thermodynamics of DNA overstretching and conformational structures of overstretched DNA that may play an important role in vivo.entropy and enthalpy | S-DNA | ssDNA | B-to-S transition

Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

“…As the ionic strength increases, there is greater electrostatic shielding within the DNA molecule, and therefore base-pair breaking is suppressed, rendering S-DNA more favorable than strand unpeeling or formation of melting bubbles. This conclusion is consistent with a number of recent studies (15,16,19). Strikingly, we find that mono-and divalent salt act differently, the latter more pronounced than expected on the basis of valence (e.g., Fig.…”

“…Instead, an alternative structure of overstretched DNA is increasingly favored as the ionic strength increases; this particular form of DNA very likely exhibits base-pairing interactions (revealed by the absence of RPA binding), although the basestacking distance and helical pitch must change to accommodate the length increase (resulting in no Sytox binding). These observations are consistent with the formation of the putative DNA structure known as S-DNA (8,11,15,16,19).…”

“…This observation substantiates several other reports that found that torsionally unconstrained dsDNA can overstretch via two distinct modes: strand unpeeling and an alternative mechanism (12,15,16,19). The latter is associated with a surprisingly small negative entropy change and is favored when intramolecular electrostatic repulsion is reduced by appropriate salt concentration and temperature (15,16,19).…”

“…In consensus with theoretical studies (8,11), it has been argued that this additional mechanism of DNA overstretching is consistent with the formation of S-DNA (4,8,11,12,19). However, as Zhang et al (16) revealed, the nature of DNA overstretching in the absence of unpeeling is complex; the competition between melting-bubble and S-DNA formation is unclear. Additionally, structural transitions associated with overstretching can also be sequencedependent, with S-DNA being favored in GC-rich sequences (19).…”

“…Three mechanisms have been proposed to account for the sudden increase in dsDNA contour length at the overstretching transition: strand unpeeling, localized base-pair breaking, and formation of S-DNA (4,5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In the unpeeling model, one strand retracts from its complementary strand via base-pair breaking.…”