Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase
TL;DR: A coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis is suggested, which further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.
About: This article is published in Cell Reports.The article was published on 2014-03-27 and is currently open access. It has received 54 citations till now. The article focuses on the topics: Helicase.
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TL;DR: The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
Abstract: A high-resolution cryo-EM structure of the heptameric Cdt1–Mcm2–7 complex of the replicative helicase from budding yeast suggests a ‘spring-action’ DNA-unwinding mechanism. The minichromosome maintenance complex (MCM) hexameric complex (Mcm2–7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1–Mcm2–7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1–MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10–15-A gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
80 citations
TL;DR: It is shown that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
63 citations
Cites background from "Single-Molecule Fluorescence Reveal..."
...…readout limits the processes that can be studied, typically requiring simplified experimental conditions with some replisome components removed and only leading-strand synthesis supported (Lee et al., 2006; Manosas et al., 2009; Pandey et al., 2009; Syed et al., 2014; Tanner et al., 2008)....
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...Such a one-dimensional readout limits the processes that can be studied, typically requiring simplified experimental conditions with some replisome components removed and only leading-strand synthesis supported (Lee et al., 2006; Manosas et al., 2009; Pandey et al., 2009; Syed et al., 2014; Tanner et al., 2008)....
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TL;DR: Techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality are reviewed.
Abstract: Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.
51 citations
TL;DR: The various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring are described.
51 citations
Cites background from "Single-Molecule Fluorescence Reveal..."
...The T7 gp4 helicase unwinds duplex DNA in 2–3 bp bursts, likely with an elementary step size of 1 nt per nucleotide hydrolysis (72)....
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TL;DR: A mechanism in which purified recombinant Drosophila melanogaster CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet is proposed, providing quantitative understanding of the central process in eukaryotic DNA replication.
Abstract: Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.
49 citations
References
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TL;DR: It is concluded that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
Abstract: The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
5,193 citations
TL;DR: Six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies remain, and a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodynamics.
Abstract: A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodynamics is presented DNA NN DG° 37 parameters from seven laboratories are presented in the same format so that careful comparisons can be made The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used dif- ferent methods of data analysis; used different salt concen- trations; and presented the NN thermodynamics in different formats As a result of these differences, there has been much confusion regarding the NN thermodynamics of DNA poly- mers and oligomers Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies re- main Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodynamics Empirical salt dependencies are also derived for oligonucleotides and polymers
2,863 citations
TL;DR: A practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes, is provided.
Abstract: Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.
1,929 citations
"Single-Molecule Fluorescence Reveal..." refers methods in this paper
...FRET values were calculated as the ratio between the acceptor intensity and the total (acceptor + donor) intensity after correcting for cross-talk between the donor and acceptor channels and subtracting the background using scripts written in Matlab (Roy et al., 2008)....
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TL;DR: This work reports the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions and shows how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence.
Abstract: We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.
1,818 citations
"Single-Molecule Fluorescence Reveal..." refers methods in this paper
...We then plotted the total unwinding rate for five different substrates and plotted it against DG/bp (dsDNA stability) calculated using the nearest-neighbor approach (Breslauer et al., 1986) and the HyTher web-based program (SantaLucia, 1998)....
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1,287 citations