The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

Abstract

It has been suggested that the structure that results when doublestranded DNA (dsDNA) is pulled from the 3 3 ends differs from that which results when it is pulled from the 5 5 ends. In this work, we demonstrate, using phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3 3 pulling versus 5 5 pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 ؎ 3 pN and 122 ؎ 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 ؎ 3 pN for 3 3 and 110 ؎ 3 pN for 5 5 ; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 ؎ 0.8 pN m for 3 3 versus 13 ؎ 8 pN for 5 5 ; and (iv) 3 3 and 5 5 molecules show different changes in hysteresis due to interactions with ␤-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA

“…Single molecule pulling experiments (SMPE) [7][8][9][10][11] and simulation studies [9,[12][13][14][15][16][17][18][19][20][21] revealed that the canonical form of double stranded DNA (B-DNA) stretches almost twice its original contour length when subjected to stretching forces. Experimental studies [7][8][9][22][23][24][25][26][27] report an overstretched transition of B-DNA when pulled with force ranging between 60 − 70 pN, and the double strands separate at sufficiently high forces [28]. A characteristic plateau has been observed in the force-extension curve of dsDNA that signifies a highly cooperative transition from B-DNA to an overstretched conformation [9].…”

“…Using constant force SMPE on λ-phage DNA, Danilowicz et al [25] reported that DNA becomes more stable with OS3 pulling than with OS5 pulling and further pointed out the differences between structures obtained from two different pulling protocols. They observed a narrow backbone spacing and rotated bases for the OS5 overstretched structure.…”

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

“…In Experiment 1, the forceresponse of overstretched DNA is inconsistent with that of one ssDNA strand or two noninteracting ssDNA strands (18,19). In Experiment 2, a second transition at an even higher force has frequently been observed that leads to final strand separation after the 65-pN overstretching transition (19)(20)(21)(22). The existence of this second transition, which is definitely a melting transition, supports the notion that the first transition (at approximately 65 pN) is not a melting transition (19)(20)(21).…”

Peeling back the mystery of DNA overstretching

“…To test this prediction, Shokri et al (16) used glyoxal modification of the DNA base pairs exposed during overstretching to show that the base pairs were indeed exposed to solution during overstretching. However, even this structural evidence was questioned as allegedly occurring because of nicks in the DNA backbone (17). Thus, despite many years of detailed study, DNA stretching experiments lacked See companion article on page 18231.…”