Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching

Tempestini, Alessia; Mónico, Carina; Gardini, Lucia; Vanzi, Francesco; Capitanio, Marco

doi:10.1093/nar/gky208

Cited by 27 publications

(26 citation statements)

References 40 publications

(65 reference statements)

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“…DNA-binding proteins employ a range of different mechanisms to interact with both select and non-select sites on DNA. 1 Key mechanistic insights have been revealed using biophysical techniques such as fluorescence microscopy, [2][3][4] optical tweezers, 5,6 surface plasmon resonance, 7 and atomic force microscopy (AFM). 8,9 AFM has been established as a powerful single-molecule technique to probe DNA-protein interactions, due to its ability to directly image DNA at nanometre resolution under physiologically relevant conditions without the need for labelling.…”

Section: Introductionmentioning

confidence: 99%

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

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Section: Introductionmentioning

confidence: 99%

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

et al. 2019

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“…A single a-catenin unfolds to bear force on actin Here, we used ultrafast force-clamp spectroscopy, a technique with sub-millisecond and sub-nanometer resolution based on laser tweezers 9,[15][16][17] , to dissect the mechanosensitivity of single mammalian a-catenin homodimers and a-b-catenin heterodimers. We first analyzed single purified recombinant a-catenin homodimers (named as a-catenin below).…”

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confidence: 99%

α-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity

Arbore

Σεργίδης

Gardini

et al. 2020

Preprint

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Cell adhesions dynamically tune their mechanical properties during tissue development and homeostasis. Fluid connections required for cell mobility can switch to solid links to maintain the mechanical rigidity of epithelial layers 1,2 . Changes in the composition and clustering of adhesion molecules have been proposed to modulate cell junction fluidity, but the underlying mechanisms are unclear 3,4 . acatenin has been shown to play a fundamental role in different adhesion sites. At adherens cell-cell junctions (AJ), a-catenin localizes in cadherin-catenin complexes, where it provides a mechanical link between b-catenin and the actin cytoskeleton 5 . However, its function is controversial owing to the low affinity between actin and the a-b-catenin heterodimer 6 . Outside AJ, a-catenin binds itself to form homodimers that connect the cell membrane to the actin cytoskeleton to promote adhesion and migration, but its mechanosensitive properties are inherently unknown 7,8 . Here, using ultra-fast laser tweezers 9 we show that a single mammalian a-catenin molecule displays very different force-bearing properties depending on whether it is associated to b-catenin or not. We found that a single a-b-catenin heterodimer slips along an actin filament in the direction of force, while a single a-catenin homodimer forms a strong asymmetric catch-bond with actin, in which the bond lifetime increases, and the protein unfolds with force. Importantly, assemblies of multiple ab-catenin heterodimers show force-bearing and unfolding properties similar to the acatenin homodimer. Our results indicate that, outside AJ, single a-catenin homodimers act as a mechanical link with the actin cytoskeleton that resists force efficiently. Nonetheless, inside AJ, a-catenin's capability to hold cell-cell connections under physiological loads critically depends on the recruitment of multiple (5-10) complexes. Our data support a molecular model in which a-catenin clustering and intercellular tension engage a fluid-to-solid phase transition at the membranecytoskeleton interface.In any living cell, an array of mechanotransductor proteins responds to mechanical cues to trigger complex molecular signaling, driving cell morphology and gene expression profiles 10 . Among these proteins, a-catenin has been reported to act as a mechanosensor that regulates adherens junctions (AJ) in response to mechanical cues from neighboring cells in a tissue 5 . It is widely accepted that a-catenin in AJ binds b-catenin, which, in turn, connects to the cytoplasmatic portion of E-cadherin to constitute the cadherin-catenin complex (CCC). Since a-catenin also binds actin, a-catenin has been indicated as a major candidate for providing a link between the CCC and the actin cytoskeleton. This molecular link is required

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“…Although 1D diffusion of Cas9 does not require external energy, we captured a surprisingly biased 1D diffusion of Cas9 on dsDNA under physiological buffer condition. Low-salt concentration, which strengthens interactions between DNA binding proteins and dsDNA (Berg et al, 1981;Bonnet et al, 2008;Lohman, 1986;Mondal and Bhattacherjee, 2015;Tempestini et al, 2018), causes Cas9 to display unbiased 1D diffusion. In addition, Cas9-VQR with altered PAM preference exhibits biased diffusion while Cas9-EQR unbiased diffuses on dsDNA under physiological salt condition (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Ionic strength is known to affect the kinetics and interactions between DNA and DNA-binding proteins (Berg et al, 1981;Bonnet et al, 2008;Lohman, 1986;Tempestini et al, 2018). To evaluate how ionic strength affects 1D diffusion of Cas9, we performed single-molecule assays shown in Fig.…”

Section: Unbiased 1d Diffusion In Low-salt Buffermentioning

confidence: 99%

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

Yang

Zhang

Sun

et al. 2019

Preprint

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The RNA-guided Streptococcus pyogenes Cas9 (spCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how the Cas9 locates its target among huge amounts of dsDNAs remains controversial. Here, combining biochemical and single-molecule assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target. We further observed a surprising biased one-dimensional diffusion of Cas9 from 3' to 5' end of the non-target strand under physiological salt condition, whereas low ionic concentration or mutations on PAM recognition residues induce unbiased one-dimensional diffusion of Cas9 along dsDNA. We quantified the diffusion length of 27 bp, which accelerates the target search efficiency of Cas9 by ~ 10 folds. Our results reveal a unique searching mechanism of Cas9 at physiological salt conditions, and provide important guidance for both in-vitro and in-vivo applications of Cas9.

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Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching

Cited by 27 publications

References 40 publications

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

α-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity

Streptococcus pyogenes Cas9 displays biased one-dimensional diffusion on dsDNA to search for a target

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