DNA polymerase activity at the single-molecule level

2011 ◽  
Vol 39 (2) ◽  
pp. 595-599 ◽  
Author(s):  
Joshua P. Gill ◽  
Jun Wang ◽  
David P. Millar
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Single Molecule Level ◽  
Nucleotide Incorporation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Conformational Rearrangements ◽  
Dna Complex

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3′→5′ exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase–DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA–protein complexes.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy

2019 ◽  
Vol 116 (23) ◽  
pp. 11247-11252 ◽  
Author(s):  
Toru Kondo ◽  
Jesse B. Gordon ◽  
Alberta Pinnola ◽  
Luca Dall’Osto ◽  
Roberto Bassi ◽  
...  
Keyword(s):  
Correlation Function ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Light Harvesting ◽  
Building Blocks ◽  
Complex Stress ◽  
Correlation Spectroscopy ◽  
Light Harvesting Complex ◽  
Single Molecule Level

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

scholarly journals Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets

2019 ◽  
Vol 47 (17) ◽  
pp. e101-e101 ◽  
Author(s):  
Boris Breiner ◽  
Kerr Johnson ◽  
Magdalena Stolarek ◽  
Ana-Luisa Silva ◽  
Aurel Negrea ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Single Molecule Detection ◽  
Synthesis Methods ◽  
Single Nucleotide ◽  
New Approach ◽  
Fluorescent Signal ◽  
Single Molecule Level ◽  
Current Sequence

AbstractA new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

scholarly journals Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Sourav Maity ◽  
Monica Mazzolini ◽  
Manuel Arcangeletti ◽  
Alejandro Valbuena ◽  
Paolo Fabris ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Force Spectroscopy ◽  
Dynamic Structure ◽  
Transmembrane Domains ◽  
Structural Studies ◽  
Single Molecule Force Spectroscopy ◽  
Conformational Rearrangements ◽  
Α Helix

Abstract Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the open state, but S3 in the closed state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

scholarly journals Imaging proteins at the single-molecule level

2017 ◽  
Vol 114 (7) ◽  
pp. 1474-1479 ◽  
Author(s):  
Jean-Nicolas Longchamp ◽  
Stephan Rauschenbach ◽  
Sabine Abb ◽  
Conrad Escher ◽  
Tatiana Latychevskaia ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Ion Beam ◽  
Low Energy ◽  
Electron Holography ◽  
Soft Landing ◽  
Single Molecule Level ◽  
Low Energy Electrons ◽  
Molecular Nanotechnology ◽  
Beam Deposition

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.

scholarly journals Optical imaging of single protein size, charge, mobility, binding and conformational change

2018 ◽  
Author(s):  
Guanzhong Ma ◽  
Hao Zhu ◽  
Zijian Wan ◽  
Yunze Yang ◽  
Shaopeng Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Near Field ◽  
Protein Analysis ◽  
Imaging Method ◽  
Charge Mobility ◽  
Single Molecule Level ◽  
Protein Size ◽  
Flexible Polymer ◽  
Protein Oscillation

AbstractProtein analysis has relied on electrophoresis, mass spectroscopy and immunoassay, which separate, detect and identify proteins based on the size, charge, mobility and binding to antibodies. However, measuring these quantities at the single molecule level has not been possible. We tether a protein to a surface with a flexible polymer, drive the protein into mechanical oscillation with an alternating electric field, and image the protein oscillation with a near field imaging method, from which we determine the size, charge, mobility of the protein. We also measure binding of antibodies to single proteins and ligand binding-induced conformational changes in single proteins. This work provides new capabilities for protein analysis and disease biomarker detection at the single molecule level.

scholarly journals Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

2019 ◽  
Vol 47 (16) ◽  
pp. 8521-8536 ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Luis M Oviedo-Bocanegra ◽  
Georg Fritz ◽  
Peter L Graumann
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Replication Forks ◽  
Single Molecule Level ◽  
Time Dynamics ◽  
Bacterium Bacillus

AbstractDNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

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