Active DNA unwinding dynamics during processive DNA replication

Abstract

Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions. In many DNA replication systems, replication and unwinding of the fork are carried out by the coordinate action of different proteins, the DNA polymerase holoenzyme and the replicative helicase, respectively (1, 2). In other systems, like the bacteriophage Phi29, these two activities are coupled within the replicative DNA polymerase (3, 4). The Phi29 DNA polymerase presents the common folding and catalytic activities characteristics of the Family B DNA polymerases (4) but in addition, it also presents an amino acid insertion in the polymerization domain, named TPR2 (Fig. S1), which confers the protein a processive strand displacement activity (5, 6). The TPR2 insertion together with the thumb, palm, and exonuclease (exo) domains forms a narrow (10 Å diameter), closed tunnel around the template strand, in comparison with the open channel described in other related DNA polymerases (5). Interestingly, this topological restriction resembles the one imposed by hexameric replicative DNA helicases at the fork junction (7, 8) and requires the dsDNA in front of the polymerase to open in order for the template to enter the active site. Therefore, the Phi29 DNA polymerase works as a hybrid polymerase-helicase and constitutes a good model system to understand the basic mechanistic principles of the coupling between DNA replication and unwinding reactions.The physical mechanism by which the polymerase replicates and promotes DNA unwinding could be described as passive; (the polymerase will not enter the duplex region until thermal fluctuations transiently open the dsDNA junction), or active (it destabilizes the duplex DNA near the junction shifting the equilibrium of the fork toward opening) (1, 9-11). The rate of a passive motor would be more sensitive than the rate of an active motor to the strength of the fork ahead (11). These two behaviors are the two extremes of a continuous spectra, and the polymerase is expected to present an unwinding mechanisms located between an ideally active and totally passive behavior (11). We used optical tweezers and a detailed kinetic analysis to probe the coupling between DNA unwinding...

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

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Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

“…Tension on the template strand of the DNA destabilizes the PTS and triggers transfer of the DNA 3 0 end to the exo site. The destabilizing effect of force on the PTS appears to be similar to that of incorporating an erroneous base, and has previously been used to study exonucleolysis by DNAp (8)(9)(10). It also has been shown that the apparent rate of polymerization goes down as tension increases up to~35 pN (8), after which DNAp seems to exclusively perform exonucleolysis (10,11).…”

Kinetic modeling of molecular motors: pause model and parameter determination from single-molecule experiments

“…Recently, we described a series of single molecules experiments designed to understand the molecular mechanism used by the Phi29 DNAp to unwind the DNA and to couple the processes of DNA replication and unwinding 5,6 . To this end, using optical tweezers, we measured the combined effect of mechanical force, favoring the DNA unwinding reaction and, DNA sequence on the real time kinetics of single polymerases molecules replicating through a DNA hairpin.…”