Tension-Dependent Stability of Torsionally Constrained DNA: Melting Precedes Overwinding

Abstract

Key genomic processes such as replication and transcription require localized unwinding and base-pair melting of the double-helix. In vivo, however, DNA is often torsionally constrained, presenting a topological barrier to unwinding. While it is known that unwinding can proceed through buckling of such DNA (resulting in plectonemes), this may only occur at low forces (typically below 1 pN). Here, we ask: how can torsionally constrained DNA unwind at higher forces without changing the overall twist?Using a combination of optical tweezers and fluorescence microscopy we map the local changes in twist and base-pair integrity of torsionally constrained DNA as a function of tension and ionic strength. By correlating the second derivatives of force-extension curves of torsionally constrained DNA to the binding of a fluorescently-labeled single-stranded DNA binding protein (Replication Protein A), we identify two tension-dependent structural transitions. In agreement with a previous proposal, we show that at high tension (above 115 pN), unwinding is accompanied by the formation of overwound DNA (termed P-DNA) in a largely cooperative mechanism. Strikingly, we also reveal that for intermediate tensions (60 to 115 pN), localized base-pair melting can occur without changes in DNA twist. This mechanism, which is significantly less cooperative, is referred to here as ‘pre-melting’. We demonstrate that pre-melting is favored by AT-rich sequences and low ionic strength. In contrast, P-DNA is largely stabilized by higher salt concentrations, while also having a preference for AT-rich domains. This supports the hypothesis that P-DNA consists of tightly entwined backbones with exposed bases.These findings provide a new understanding of the interplay between DNA twist, extension and sequence. Moreover, since DNA unwinding can be induced through either tension or applied torque, this work may have strong implications for the metabolism of torsionally constrained DNA in vivo.