Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. The present single-molecule based approach complements high-resolution structural methods in deciphering the molecular mechanisms of the helicases.
The dynamic interactions between the exposed nucleotides and the helicases underlay the force- and salt-dependences of their enzymatic activities. The synergetic approach reveals that the interactions between the exposed nucleotides and the helicases could be reduced by large stretching forces or electrostatically shielded with high-concentration salt, subsequently resulting in reduced translocation rates of the helicases. The whole nucleotide segment possessed curved conformations and covered the two RecA-like domains of the helicases, which are essential for the inch-worm mechanism. Taking two Pif1 helicases (ScPif1 and BsPif1) as model systems, we found that, besides a few tightly bound nucleotides, adjacent solvent-exposed nucleotides interact dynamically with the helicase surfaces. In this study, we showed that single-molecule techniques, in combination with computational modeling, can characterize dynamic conformations not resolved by high-resolution structure determination methods. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.įlexible regions in biomolecular complexes, although crucial to understanding structure–function relationships, are often unclear in high-resolution crystal structures. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. The strategy improved the resolution of Förster resonance energy transfer to 0.5 bp, high enough to uncover differences in DNA unwinding by yeast Pif1 and E. We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs, just as in optical or magnetic tweezers. However, it has been lacking single-base pair (1-bp) resolution required for revealing stepping kinetics of helicases.
Single-molecule Förster resonance energy transfer is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA.
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