scholarly journals Modelling single-molecule kinetics of helicase translocation using high-resolution nanopore tweezers (SPRNT)

2021 ◽  
Author(s):  
Jonathan M. Craig ◽  
Andrew H. Laszlo ◽  
Ian C. Nova ◽  
Jens H. Gundlach
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Specific Enzyme ◽  
Spatiotemporal Resolution ◽  
Single Stranded Dna ◽  
Detailed Model ◽  
Specific Location ◽  
Dna Strand ◽  
Kinetics Of ◽  
Dna Substrate

Abstract Single-molecule picometer resolution nanopore tweezers (SPRNT) is a technique for monitoring the motion of individual enzymes along a nucleic acid template at unprecedented spatiotemporal resolution. We review the development of SPRNT and the application of single-molecule kinetics theory to SPRNT data to develop a detailed model of helicase motion along a single-stranded DNA substrate. In this review, we present three examples of questions SPRNT can answer in the context of the Superfamily 2 helicase Hel308. With Hel308, SPRNT’s spatiotemporal resolution enables resolution of two distinct enzymatic substates, one which is dependent upon ATP concentration and one which is ATP independent. By analyzing dwell-time distributions and helicase back-stepping, we show, in detail, how SPRNT can be used to determine the nature of these observed steps. We use dwell-time distributions to discern between three different possible models of helicase backstepping. We conclude by using SPRNT’s ability to discern an enzyme’s nucleotide-specific location along a DNA strand to understand the nature of sequence-specific enzyme kinetics and show that the sequence within the helicase itself affects both step dwell-time and backstepping probability while translocating on single-stranded DNA.

scholarly journals Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

2019 ◽  
Author(s):  
Sungchul Kim ◽  
Luuk Loeff ◽  
Sabina Colombo ◽  
Stan J.J. Brouns ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Comprehensive Understanding ◽  
Single Molecule Fluorescence ◽  
Spatiotemporal Resolution ◽  
Single Stranded Dna ◽  
Correct Orientation ◽  
Crispr Array ◽  
Cellular Dna

AbstractCRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

scholarly journals First passage time study of DNA strand displacement

2020 ◽  
Author(s):  
D. W. Bo Broadwater ◽  
Alexander W. Cook ◽  
Harold D. Kim
Keyword(s):  
Single Molecule ◽  
First Passage Time ◽  
Resonance Energy ◽  
Passage Time ◽  
Single Step ◽  
Strand Displacement ◽  
First Passage ◽  
Dna Strand Displacement ◽  
Dna Strand ◽  
Kinetics Of

AbstractDNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) approach termed the “fission” assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence except for T→U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ∼30 µs to ∼300 µs depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

scholarly journals Direct Observation of Topoisomerase IA Gate Dynamics

2018 ◽  
Author(s):  
Maria Mills ◽  
Yuk-Ching Tse-Dinh ◽  
Keir C. Neuman
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Mechanistic Model ◽  
Cellular Functions ◽  
Single Stranded Dna ◽  
Gate Opening ◽  
Type Ia ◽  
Dna Strand ◽  
Dynamics Simulations ◽  
Strand Passage

AbstractType IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

scholarly journals Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers

2017 ◽  
Vol 114 (45) ◽  
pp. 11932-11937 ◽  
Author(s):  
Jonathan M. Craig ◽  
Andrew H. Laszlo ◽  
Henry Brinkerhoff ◽  
Ian M. Derrington ◽  
Matthew T. Noakes ◽  
...  
Keyword(s):  
High Resolution ◽  
Kinetic Model ◽  
Dna Sequence ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Single Stranded Dna ◽  
The Core ◽  
Detailed Kinetic Model ◽  
Kinetics Of

Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.

scholarly journals Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

2020 ◽  
Vol 21 (19) ◽  
pp. 7389
Author(s):  
Aleksandr Alekseev ◽  
Galina Cherevatenko ◽  
Maksim Serdakov ◽  
Georgii Pobegalov ◽  
Alexander Yakimov ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Strand Exchange ◽  
Single Stranded Dna ◽  
Dna Strand Exchange ◽  
High Conservation ◽  
Dna Strand

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

scholarly journals Monitoring G-quadruplex formation with DNA carriers and solid-state nanopores

2019 ◽  
Author(s):  
Filip Bošković ◽  
Jinbo Zhu ◽  
Kaikai Chen ◽  
Ulrich F. Keyser
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Drug Targets ◽  
Gene Expression Regulation ◽  
Single Stranded Dna ◽  
Single Molecule Level ◽  
G Quadruplex ◽  
Human Viruses ◽  
Kinetics Of ◽  
Dna Carriers

ABSTRACTG-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ~5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un-)folded Gq structures, provides insights into single-molecule kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-molecule level without labelling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.

scholarly journals Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids

2017 ◽  
Vol 121 (12) ◽  
pp. 2594-2602 ◽  
Author(s):  
Xiaoping Olson ◽  
Shohei Kotani ◽  
Bernard Yurke ◽  
Elton Graugnard ◽  
William L. Hughes
Keyword(s):  
Nucleic Acids ◽  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Locked Nucleic Acids ◽  
Dna Strand ◽  
Kinetics Of

On the use of Ozawa/Flynn/Wall method to determine aging activation energy for elastomers

2021 ◽  
pp. 009524432110203
Author(s):  
Sudhir Bafna
Keyword(s):  
Mechanical Properties ◽  
Activation Energy ◽  
Weight Loss ◽  
Oxygen Consumption ◽  
Dwell Time ◽  
Room Temperature ◽  
Elevated Temperatures ◽  
Degradation Mechanism ◽  
Kinetics Of ◽  
Wall Method

It is often necessary to assess the effect of aging at room temperature over years/decades for hardware containing elastomeric components such as oring seals or shock isolators. In order to determine this effect, accelerated oven aging at elevated temperatures is pursued. When doing so, it is vital that the degradation mechanism still be representative of that prevalent at room temperature. This places an upper limit on the elevated oven temperature, which in turn, increases the dwell time in the oven. As a result, the oven dwell time can run into months, if not years, something that is not realistically feasible due to resource/schedule constraints in industry. Measuring activation energy (Ea) of elastomer aging by test methods such as tensile strength or elongation, compression set, modulus, oxygen consumption, etc. is expensive and time consuming. Use of kinetics of weight loss by ThermoGravimetric Analysis (TGA) using the Ozawa/Flynn/Wall method per ASTM E1641 is an attractive option (especially due to the availability of commercial instrumentation with software to make the required measurements and calculations) and is widely used. There is no fundamental scientific reason why the kinetics of weight loss at elevated temperatures should correlate to the kinetics of loss of mechanical properties over years/decades at room temperature. Ea obtained by high temperature weight loss is almost always significantly higher than that obtained by measurements of mechanical properties or oxygen consumption over extended periods at much lower temperatures. In this paper, data on five different elastomer types (butyl, nitrile, EPDM, polychloroprene and fluorocarbon) are presented to prove that point. Thus, use of Ea determined by weight loss by TGA tends to give unrealistically high values, which in turn, will lead to incorrectly high predictions of storage life at room temperature.

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

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