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

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.

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The Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore

10.26434/chemrxiv.6383213.v1 ◽

2018 ◽

Author(s):

Xin Shi ◽

Qiao Li ◽

Rui Gao ◽

Wei Si ◽

Shao-Chuang Liu ◽

...

Keyword(s):

Solid State ◽

Single Molecule ◽

Conformational Changes ◽

Ionic Current ◽

Observation Time ◽

Random Coil ◽

Dna Complexes ◽

Dna Complex ◽

Identification And Characterization

<a></a><a>Docking of a protein-DNA complex onto a nanopore can provide ample observation time for a detailed inspection of the complex, enabling collection of biophysical data for detection, identification, and characterization of the biomolecules. While docking of a protein-DNA complex onto a biological nanopore has enabled analytic applications of nanopores including DNA sequencing, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behaviour of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. </a><a>Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels within the same current blockade. </a>We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels whereas the frequency of the current level switching to be determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif states.

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The Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore

10.26434/chemrxiv.6383213 ◽

2018 ◽

Author(s):

Xin Shi ◽

Qiao Li ◽

Rui Gao ◽

Wei Si ◽

Shao-Chuang Liu ◽

...

Keyword(s):

Solid State ◽

Single Molecule ◽

Conformational Changes ◽

Ionic Current ◽

Observation Time ◽

Random Coil ◽

Dna Complexes ◽

Dna Complex ◽

Identification And Characterization

<a></a><a>Docking of a protein-DNA complex onto a nanopore can provide ample observation time for a detailed inspection of the complex, enabling collection of biophysical data for detection, identification, and characterization of the biomolecules. While docking of a protein-DNA complex onto a biological nanopore has enabled analytic applications of nanopores including DNA sequencing, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behaviour of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. </a><a>Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels within the same current blockade. </a>We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels whereas the frequency of the current level switching to be determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif states.

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DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

10.1101/306175 ◽

2018 ◽

Author(s):

Carel Fijen ◽

Mariam Mahmoud ◽

Rebecca Kaup ◽

Jamie Towle-Weicksel ◽

Joann Sweasy ◽

...

Keyword(s):

Dna Repair ◽

Dna Polymerase ◽

Single Molecule ◽

Conformational Changes ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dna Polymerase Β ◽

Gapped Dna ◽

Dna Substrate

The eukaryotic DNA polymerase β plays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA 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 substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

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Real-time analysis of RAG complex activity in V(D)J recombination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606721113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11853-11858 ◽

Cited By ~ 9

Author(s):

Jennifer Zagelbaum ◽

Noriko Shimazaki ◽

Zitadel Anne Esguerra ◽

Go Watanabe ◽

Michael R. Lieber ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Conformational Changes ◽

Signal Sequence ◽

High Mobility ◽

Time Analysis ◽

Complex Activity ◽

Synaptic Complex ◽

Single Molecule Fret ◽

Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

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Probing Conformational Changes and Dynamics in eIF4A Helicase during RNA Unwinding by Single-Molecule FRET

Biophysical Journal ◽

10.1016/j.bpj.2012.11.2345 ◽

2013 ◽

Vol 104 (2) ◽

pp. 421a

Author(s):

Yingjie Sun ◽

Amit Meller

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Single Molecule Fret ◽

Rna Unwinding

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Single-Molecule FRET Analysis of Protein-DNA Complexes

Methods in Molecular Biology™ - DNA-Protein Interactions ◽

10.1007/978-1-60327-015-1_29 ◽

2009 ◽

pp. 503-521 ◽

Cited By ~ 8

Author(s):

Mike Heilemann ◽

Ling Chin Hwang ◽

Konstantinos Lymperopoulos ◽

Achillefs N. Kapanidis

Keyword(s):

Single Molecule ◽

Dna Complexes ◽

Single Molecule Fret

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Mechanistic insights into Lhr helicase function in DNA repair

Biochemical Journal ◽

10.1042/bcj20200379 ◽

2020 ◽

Vol 477 (16) ◽

pp. 2935-2947

Author(s):

Ryan J. Buckley ◽

Kevin Kramm ◽

Christopher D. O. Cooper ◽

Dina Grohmann ◽

Edward L. Bolt

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Single Molecule ◽

Dna Helicase ◽

Holliday Junctions ◽

Single Molecule Fret ◽

Repair Enzymes ◽

Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

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