Molecular Force Balance Measurements Reveal that Double-Stranded DNA Unbinds Under Force in Rate-Dependent Pathways

Strand separation of double-stranded DNA is a crucial step for essential cellular processes such as recombination and transcription. By means of a molecular force balance, we have analyzed the impact of different pulling directions and different force-loading rates on the unbinding process of short double-stranded DNA. At loading rates above 9 × 105 pN/s, we found a marked difference in rupture probability for pulling the duplex in 3′-3′ direction compared to a 5′-5′ direction, indicating different unbinding pathways. We propose a mechanism by which unbinding at low loading rates is dominated by nondirectional thermal fluctuations, whereas mechanical properties of the DNA become more important at high loading rates and reveal the asymmetry of the phosphoribose backbone. Our model explains the difference of 3′-3′ and 5′-5′ unbinding as a kinetic process, where the loading rate exceeds the relaxation time of DNA melting bubbles.