scholarly journals Acceleration of DNA Replication of Klenow Fragment by Small Resisting Force

2021 ◽  
Vol 38 (11) ◽  
pp. 118701
Author(s):  
Yu-Ru Liu ◽  
Peng-Ye Wang ◽  
Wei Li ◽  
Ping Xie
Keyword(s):  
Dna Replication ◽  
External Force ◽  
Single Molecule ◽  
Molecular Motors ◽  
Dna Polymerases ◽  
Maximum Velocity ◽  
Magnetic Tweezers ◽  
Coupling Mechanism ◽  
Klenow Fragment ◽  
Chemomechanical Coupling

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force, attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.

scholarly journals A model of processive walking and slipping of kinesin-8 molecular motors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ping Xie
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
External Load ◽  
Molecular Motor ◽  
Velocity Versus ◽  
Stick Slip ◽  
Load Dependence ◽  
Chemomechanical Coupling ◽  
Similarities And Differences ◽  
Stick Slip Motion

AbstractKinesin-8 molecular motor can move with superprocessivity on microtubules towards the plus end by hydrolyzing ATP molecules, depolymerizing microtubules. The available single molecule data for yeast kinesin-8 (Kip3) motor showed that its superprocessive movement is frequently interrupted by brief stick–slip motion. Here, a model is presented for the chemomechanical coupling of the kinesin-8 motor. On the basis of the model, the dynamics of Kip3 motor is studied analytically. The analytical results reproduce quantitatively the available single molecule data on velocity without including the slip and that with including the slip versus external load at saturating ATP as well as slipping velocity versus external load at saturating ADP and no ATP. Predicted results on load dependence of stepping ratio at saturating ATP and load dependence of velocity at non-saturating ATP are provided. Similarities and differences between dynamics of kinesin-8 and that of kinesin-1 are discussed.

scholarly journals A Generalized Kinetic Model for Coupling between Stepping and ATP Hydrolysis of Kinesin Molecular Motors

2019 ◽  
Vol 20 (19) ◽  
pp. 4911 ◽  
Author(s):  
Xie ◽  
Guo ◽  
Chen
Keyword(s):  
Kinetic Model ◽  
Atpase Activity ◽  
Single Molecule ◽  
Rate Constants ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Power Production ◽  
Wild Type ◽  
Chemomechanical Coupling ◽  
Generalized Kinetic Model

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.

scholarly journals Two essential replicative DNA polymerases exchange dynamically during DNA replication and after replication fork arrest

2018 ◽  
Author(s):  
Yilai Li ◽  
Ziyuan Chen ◽  
Lindsay A. Matthews ◽  
Lyle A. Simmons ◽  
Julie S. Biteen
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Super Resolution ◽  
Clamp Loader ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Replication Process ◽  
Cooperative Process

AbstractThe replisome is the multi-protein complex responsible for faithful replication of chromosomal DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in liveBacillus subtiliscells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following both damage-independent and damage-dependent replication fork stress. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replisomal proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process, and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results show that DnaE provides a limited contribution to chromosomal replication and repair.

scholarly journals Single-Molecule Insights Into the Dynamics of Replicative Helicases

2021 ◽  
Vol 8 ◽  
Author(s):  
Richard R. Spinks ◽  
Lisanne M. Spenkelink ◽  
Nicholas E. Dixon ◽  
Antoine M. van Oijen
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Replication Fork ◽  
Magnetic Tweezers ◽  
Replication Initiation ◽  
Model Systems ◽  
Replication Forks ◽  
Nucleotide Hydrolysis ◽  
Replicative Polymerases ◽  
Replication Factors

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. 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. 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. 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. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

scholarly journals 3P170 Single molecule dynamics of the epsilon subunit in F_1 forced-rotated by magnetic tweezers(Molecular motors,Oral Presentations)

2007 ◽  
Vol 47 (supplement) ◽  
pp. S245
Author(s):  
Eiichiro Saita ◽  
Ryota Iino ◽  
Yasuyuki Yamada-Kato ◽  
Toshiharu Suzuki ◽  
Hiroyuki Noji ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Magnetic Tweezers ◽  
Epsilon Subunit ◽  
Single Molecule Dynamics ◽  
Oral Presentations ◽  
Molecule Dynamics

scholarly journals Single-molecule FRET dynamics of molecular motors in an ABEL Trap

2020 ◽  
Author(s):  
Maria Dienerowitz ◽  
Jamieson A. L. Howard ◽  
Steven D. Quinn ◽  
Frank Dienerowitz ◽  
Mark C. Leake
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Rational Design ◽  
Molecular Motor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Observation Time

AbstractSingle-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap’s inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. Here we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with 1 ms time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

Studies of DNA-Replication at the Single Molecule Level Using Magnetic Tweezers

2010 ◽  
pp. 89-122
Author(s):  
Maria Manosas ◽  
Timothée Lionnet ◽  
Élise Praly ◽  
Ding Fangyuan ◽  
Jean-François Allemand ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Single Molecule Level

scholarly journals Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

2010 ◽  
Vol 107 (5) ◽  
pp. 1900-1905 ◽  
Author(s):  
Candice M. Etson ◽  
Samir M. Hamdan ◽  
Charles C. Richardson ◽  
Antoine M. van Oijen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Model System ◽  
Double Stranded Dna ◽  
Nucleotide Incorporation ◽  
Imaging Approach ◽  
Processivity Factor

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

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