scispace - formally typeset
Search or ask a question
Author

Marie-Noëlle Dessinges

Bio: Marie-Noëlle Dessinges is an academic researcher from Centre national de la recherche scientifique. The author has contributed to research in topics: Processivity & Replisome. The author has an hindex of 1, co-authored 1 publications receiving 179 citations.

Papers
More filters
Journal ArticleDOI
TL;DR: A single-molecule manipulation technique is used to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme, and observes a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands.
Abstract: DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3′-5′ on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.

189 citations


Cited by
More filters
Journal ArticleDOI
TL;DR: Through structural and single-molecule investigations, researchers are developing coherent models to explain the functions and dynamic motions of helicase enzymes.
Abstract: Helicases and remodeling enzymes are ATP-dependent motor proteins that play a critical role in every aspect of RNA and DNA metabolism. Most RNA-remodeling enzymes are members of helicase superfamily 2 (SF2), which includes many DNA helicase enzymes that display similar structural and mechanistic features. Although SF2 enzymes are typically called helicases, many of them display other types of functions, including single-strand translocation, strand annealing, and protein displacement. There are two mechanisms by which RNA helicase enzymes unwind RNA: The nonprocessive DEAD group catalyzes local unwinding of short duplexes adjacent to their binding sites. Members of the processive DExH group often translocate along single-stranded RNA and displace paired strands (or proteins) in their path. In the latter case, unwinding is likely to occur by an active mechanism that involves Brownian motor function and stepwise translocation along RNA. Through structural and single-molecule investigations, researchers are developing coherent models to explain the functions and dynamic motions of helicase enzymes.

451 citations

Journal ArticleDOI
TL;DR: This review discusses single-molecule experiments (SMEs) in biological physics from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied.
Abstract: I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

386 citations

Journal ArticleDOI
05 Jan 2006-Nature
TL;DR: This work follows in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer, a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target.
Abstract: Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.

359 citations

Journal ArticleDOI
TL;DR: Self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities, and the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity.
Abstract: Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.

325 citations