13,461 research outputs found
Unwinding biological systems
Unwinding conditions have been fruitfully exploited in Information Flow Security to define persistent security properties. In this paper we investigate their meaning and possible uses in the analysis of biological systems. In particular, we elaborate on the notion of robustness and propose some instances of unwinding over the process algebra Bio-PEPA and over hybrid automata. We exploit such instances to analyse two case-studies: Neurospora crassa circadian system and Influenza kinetics models
Velocity and processivity of helicase unwinding of double-stranded nucleic acids
Helicases are molecular motors which unwind double-stranded nucleic acids
(dsNA) in cells. Many helicases move with directional bias on single-stranded
(ss) nucleic acids, and couple their directional translocation to strand
separation. A model of the coupling between translocation and unwinding uses an
interaction potential to represent passive and active helicase mechanisms. A
passive helicase must wait for thermal fluctuations to open dsNA base pairs
before it can advance and inhibit NA closing. An active helicase directly
destabilizes dsNA base pairs, accelerating the opening rate. Here we extend
this model to include helicase unbinding from the nucleic-acid strand. The
helicase processivity depends on the form of the interaction potential. A
passive helicase has a mean attachment time which does not change between ss
translocation and ds unwinding, while an active helicase in general shows a
decrease in attachment time during unwinding relative to ss translocation. In
addition, we describe how helicase unwinding velocity and processivity vary if
the base-pair binding free energy is changed.Comment: To appear in special issue on molecular motors, Journal of Physics -
Condensed Matte
A two-state model for helicase translocation and unwinding of nucleic acids
Helicases are molecular motors that unwind double-stranded nucleic acids
(dsNA), such as DNA and RNA). Typically a helicase translocates along one of
the NA single strands while unwinding and uses adenosine triphosphate (ATP)
hydrolysis as an energy source. Here we model of a helicase motor that can
switch between two states, which could represent two different points in the
ATP hydrolysis cycle. Our model is an extension of the earlier
Betterton-J\"ulicher model of helicases to incorporate switching between two
states. The main predictions of the model are the speed of unwinding of the
dsNA and fluctuations around the average unwinding velocity. Motivated by a
recent claim that the NS3 helicase of Hepatitis C virus follows a flashing
ratchet mechanism, we have compared the experimental results for the NS3
helicase with a special limit of our model which corresponds to the flashing
ratchet scenario. Our model accounts for one key feature of the experimental
data on NS3 helicase. However, contradictory observations in experiments
carried out under different conditions limit the ability to compare the model
to experiments.Comment: minor modification
Coupling of Two Motor Proteins: a New Motor Can Move Faster
We study the effect of a coupling between two motor domains in
highly-processive motor protein complexes. A simple stochastic discrete model,
in which the two parts of the protein molecule interact through some energy
potential, is presented. The exact analytical solutions for the dynamic
properties of the combined motor species, such as the velocity and dispersion,
are derived in terms of the properties of free individual motor domains and the
interaction potential. It is shown that the coupling between the motor domains
can create a more efficient motor protein that can move faster than individual
particles. The results are applied to analyze the motion of helicase RecBCD
molecules
Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent \u3cem\u3ePlasmodium falciparum\u3c/em\u3e SSB as a Reporter for Single-Stranded DNA
Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism
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Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.
The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling
DNA structure
Deoxyribonucleic acid (DNA) is a polymer of nucleotides. In the cell, DNA usually adopts a double-stranded helical form, with complementary base-pairing holding the two strands together. The most stable conformation is called B-form DNA, although other structures can occur under specific conditions
Homologous Recombination under the Single-Molecule Fluorescence Microscope
Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination
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