Single-Molecule Studies of the E. Coli Translesion Replisome

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

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Single-molecule studies contrast ordered DNA replication with stochastic translesion synthesis

eLife ◽

10.7554/elife.32177 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 15

Author(s):

Gengjing Zhao ◽

Emma S Gleave ◽

Meindert Hugo Lamers

Keyword(s):

Dna Adducts ◽

Single Molecule ◽

Uv Light ◽

Dna Polymerases ◽

Stable Complex ◽

High Fidelity ◽

Single Molecule Spectroscopy ◽

E Coli ◽

Single Molecule Studies ◽

Translesion Polymerases

High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793522148 ◽

2016 ◽

Author(s):

Olivier Hyrien

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793506614 ◽

2015 ◽

Author(s):

Antoine van Oijen

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

International Journal of Molecular Sciences ◽

10.3390/ijms22052398 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2398

Author(s):

Wooyoung Kang ◽

Seungha Hwang ◽

Jin Young Kang ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Facilitated Diffusion ◽

One Dimensional ◽

Bacterial Transcription ◽

Fluorescence Measurements ◽

Dimensional Diffusion ◽

Single Molecule Studies ◽

Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

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Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

Australian Journal of Chemistry ◽

10.1071/ch09615 ◽

2010 ◽

Vol 63 (4) ◽

pp. 624

Author(s):

Michael J. Serpe ◽

Jason R. Whitehead ◽

Stephen L. Craig

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Monomer Concentration ◽

Supramolecular Polymer ◽

Force Microscopy ◽

Molecular Weight Distributions ◽

Atomic Force ◽

Multi Stage ◽

Single Molecule Studies ◽

Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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