Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
Bottom Line: To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication.According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state.We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).
Affiliation: Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.Show MeSH
Related in: MedlinePlus
Mentions: Instead, our data is compatible with a loose-coupling model between chemical catalysis and mechanical translocation in which translocation occurs following the PPi release and before dNTP binding steps (Figure 1D). The results from the fits rendered an associated average free energy change for translocation, ΔGtrans = −0.46kBT, and an effective translocation step size, δ = 0.40 nm. These results support an energy landscape for translocation where the polymerase-DNA complex could thermally diffuse between pre- and post- translocated states separated by a distance equivalent to the mean rise per base found in B-DNA, spending ∼1.6 more time in the post-translocated state (Figure 5). The different distances associated with the forward and backward translocation rates, dT = 0.35 nm and d−T = 0.05 nm, respectively, indicate that the transition state barrier is very close to post-translocation state (Figure 5). The effect of opposing load is to shift the translocation equilibrium mainly by slowing the forward translocation rate, kT(0), or the exit from pre- to the post-translocated state. The similarity we found between the force sensitive forward translocation rate and the rate limiting step of the reaction, kcat [kT(0) is only five times faster than kcat, Table 1], explains naturally the marked sensitivity of replication velocity to load even at saturated dNTP concentrations (Figure 3A). Therefore, the force-dependent translocation and catalysis control the overall replication velocity and the force–velocity relationship. A similar observation has been recently reported from single molecule mechano-chemical studies of the RNA polymerase II (35). We note that the activity of the Phi29 DNAP is not affected by mechanical tension below ∼20 pN applied longitudinally to the DNA (41–43). This observation indicates that DNA mechanical tension, which is expected to build on the DNA as load is increased on the replicative complex, cannot be responsible for the velocity changes measured even at the smallest aiding or opposing forces. Additional force dependencies on different steps of the replication cycle were not required to fit the experimental data (Supplementary Data), arguing against other possible additional effects of load on the nucleotide incorporation cycle or on the protein activity.
Affiliation: Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, 28049 Madrid, Spain.