Site-Search Process for Synaptic Protein-DNA Complexes

Vemulapalli, Sridhar; Hashemi, Mohtadin; Lyubchenko, Yuri L.

doi:10.3390/ijms23010212

Cited by 8 publications

(27 citation statements)

References 28 publications

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“…Time-lapse AFM imaging is highly attractive for biological studies, as it allows one to perform imaging close to physiological conditions by avoiding sample drying [ 27 , 28 ]. The use of HS-AFM instrumentation is especially attractive, as it will enable the direct visualization of dynamics at a high data acquisition rate [ 29 , 30 , 31 ]. We demonstrated here that labeling with 3WJ is fully compatible with such AFM instrumentation.…”

Section: Discussionmentioning

confidence: 99%

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Sun

Stormberg

Filliaux

et al. 2022

IJMS

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Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

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Section: Discussionmentioning

confidence: 99%

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Sun

Stormberg

Filliaux

et al. 2022

IJMS

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“…A total of 9 DNA duplexes with and without the specific site (GGCCTCGAGG GCC) for SfiI were designed with either 23 bp or 33 bp lengths, as shown in Figure S1. The DNA sequences for the duplex were taken from the 3-site DNA substrate used in our previous studies (20,21). Briefly, DNA oligonucleotides, with and without the SfiI specific site and a thiol modification at the 3' end, were purchased from Integrated DNA Technologies (Coralville, IA) as singlestranded complements.…”

Section: Preparation Of Dna Duplexesmentioning

confidence: 99%

“…However, the model is limited to the site search process for a single site and is not applicable to proteins interacting with multiple specific sites on DNA. To understand these long-range interactions on DNA, we have previously used the restriction enzyme SfiI as a model system, which cleaves DNA after binding to two cognate sites using Mg 2+ cations as cofactors (9,(16)(17)(18)(19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

“…Assembly of a synaptic complex with the DNA molecules containing two cognate sites results in looped DNA complexes with the loop sizes corresponding to the distances between the two cognate sites (9,10,18). In our recent studies, we used time-lapse HS-AFM to directly visualize the dynamics of the site search process by SfiI (21). Using this approach, we visualized sliding and jumping pathways leading to the SfiI binding to DNA.…”

Section: Introductionmentioning

confidence: 99%

“…In another pathway, the complex of SfiI bound to the specific sites jumps over a large distance forming a looped complex. These studies led to the proposed site search model for SfiI (21). According to this model, the search for the first specific site can occur via the BHW model, followed by the formation of synaptic complexes via the new pathways described above.…”

Section: Introductionmentioning

confidence: 99%

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Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions

Vemulapalli

Hashemi

Kolomeisky

et al. 2023

Preprint

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The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, there was an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates has been found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment is developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

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Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action

2023

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High-speed atomic force microscopy (HS-AFM) is a unique approach that allows direct real-time visualization of biological macromolecules in action under near-physiological conditions, without any chemical labeling. Typically, the temporal resolution is sub-100 ms, and the spatial resolution is 2–3 nm in the lateral direction and ~0.1 nm in the vertical direction. A wide range of biomolecular systems and their dynamic processes have been studied by HS-AFM, providing deep mechanistic insights into how biomolecules function. However, the level of mechanistic detail gleaned from an HS-AFM experiment critically depends on the spatiotemporal resolution of the system. In this review article, we explain the principle of HS-AFM, and describe how the resolution is determined. We also discuss recent attempts to improve the resolution of HS-AFM to further extend the observable range of biological phenomena.

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Site-Search Process for Synaptic Protein-DNA Complexes

Cited by 8 publications

References 28 publications

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions

Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action

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