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. Continued abuse of our services will cause your IP address to be blocked indefinitely.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. Please fill out the CAPTCHA below and then click the button to indicate that you agree to these terms. If you wish to be unblocked, you must agree that you will take immediate steps to rectify this issue. If you do not understand what is causing this behavior, please contact us here. If you promise to stop (by clicking the Agree button below), we'll unblock your connection for now, but we will immediately re-block it if we detect additional bad behavior. Overusing our search engine with a very large number of searches in a very short amount of time.Using a badly configured (or badly written) browser add-on for blocking content.Running a "scraper" or "downloader" program that either does not identify itself or uses fake headers to elude detection.Using a script or add-on that scans GameFAQs for box and screen images (such as an emulator front-end), while overloading our search engine. ![]() There is no official GameFAQs app, and we do not support nor have any contact with the makers of these unofficial apps. Continued use of these apps may cause your IP to be blocked indefinitely. ![]() This triggers our anti-spambot measures, which are designed to stop automated systems from flooding the site with traffic. ![]()
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