Abstract
DNA polymerase couples chemical energy to translocation along a DNA template with a specific directionality while it replicates genetic information. According to single-molecule manipulation experiments, the polymerase-DNA complex can work against loads greater than 50 pN. It is not known, on the one hand, how chemical energy is transduced into mechanical motion, accounting for such large forces on sub-nanometer steps, and, on the other hand, how energy consumption in fidelity maintenance integrates in this non-equilibrium cycle. Here, we propose a translocation mechanism that points to the flexibility of the DNA, including its overstretching transition, as the principal responsible for the DNA polymerase ratcheting motion. By using thermodynamic analyses, we then find that an external load hardly affects the fidelity of the copying process and, consequently, that translocation and fidelity maintenance are loosely coupled processes. The proposed translocation mechanism is compatible with single-molecule experiments, structural data and stereochemical details of the DNA-protein complex that is formed during replication, and may be extended to RNA transcription.
Highlights
A polymerase is a motor protein that transfers genetic information inside biological cells
ATP binding to the forward head attaches this head to the microtubule track and ATP hydrolysis in the rear head releases this head from the microtubule
We propose the following DNA-centered translocation mechanism in DNA replication: Soon after the new nucleotide is branched to the replicated strand, the newly formed base-pair experiences a hydrophobic interaction with the previous base-pair, triggering a conformational change
Summary
We have performed a thermodynamic analysis of the single-nucleotide addition cycle in DNA replication, the first that conjugates mechanical and information aspects. We propose that the DNA elasticity has a central role in the translocation of the DNAp relative to the DNA substrate This includes the overstretchig transition, which associated DNA structure change at high force are observed as the reverse of the base-pairing and base-stacking processes that take place during nucleotide incorporation in DNA replication. High opposing loads below the overstretching transition force do not abruptly change the DNA rise per base-pair and the base-stacking process of the nascent base-pairs. These forces, while hindering and slowing the DNAp, do not necessarily stall the protein activity, as experimentaly found[28]. These changes are, besides, protein-dependent, as needed to account for the diverse fidelities and structural details of the different polymerases
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