Transient Kinetics Measured with Force Steps Discriminate between Double Stranded DNA Elongation and Melting and Define the Reaction Energetics

Abstract

Double stranded DNA pulled to ∼65 pN undergoes an overstretching transition from the basic conformation (B-form) to a 1.7 times longer conformation that represents a fundamental stage in the structural transitions during DNA recombination, replication and repair. By using a dual laser optical tweezers with a fast force feedback (2 ms rise time), we recorded the length transient following force steps imposed on the λ-phage DNA with different degrees of melting and at different temperatures (10-25°C). The rate-force relations obtained from the lengthening transient under our rapid force clamp following 2-35 pN force pull to the overstretching force shows that the whole 70% extension is a two state reaction from the B-form to an S-form that precedes and is independent of melting. The shortening transient following 20-35 pN force drop from the overstretching force is made by stepwise shortenings and pauses due to S-B transitions and reannealing of the melted segments. The temperature dependence of the lengthening transient shows that the entropic contribution to the reaction at room temperature is only 1/3 of the entropy change expected from thermal melting. The structural parameters that describe the distances from the B and the S state to the transition state, do not depend on temperature. Accordingly the total elongation of a single two-state unit, and therefore q, the index of cooperativity (22bp) of the reaction, do not depend on temperature, suggesting that it arises from structural factors, such as the nucleic acid sequence. Supported by IIT-SEED (Genova) and Ente Cassa di Risparmio di Firenze.