scholarly journals Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

2022 ◽  
Vol 5 (4) ◽  
pp. e202101078
Author(s):  
Tunahan Ergünay ◽  
Özgecan Ayhan ◽  
Arda B Celen ◽  
Panagiota Georgiadou ◽  
Emre Pekbilir ◽  
...  
Keyword(s):  
Dna Binding ◽  
Regulatory Mechanisms ◽  
Clinical Implementation ◽  
Post Translational Modifications ◽  
Guide Rna ◽  
Nuclease Domain ◽  
Target Dna ◽  
Unwanted Consequence ◽  
Positively Charged ◽  
Pharmacologic Inhibition

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

scholarly journals Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zhicheng Zuo ◽  
Ashwini Zolekar ◽  
Kesavan Babu ◽  
Victor JT Lin ◽  
Hamed S Hayatshahi ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Dna Cleavage ◽  
Ternary Complex ◽  
Catalytic Triad ◽  
Guide Rna ◽  
Nuclease Domain ◽  
Target Dna ◽  
Bona Fide ◽  
Experimental Approaches ◽  
Dna Strand

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.

scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

Equilibrium shift by target DNA substrates for determination of DNA binding ligands

2007 ◽  
Vol 17 (1) ◽  
pp. 68-72 ◽  
Author(s):  
Saori Tsujita ◽  
Mikimasa Tanada ◽  
Tomonobu Kataoka ◽  
Shigeki Sasaki
Keyword(s):  
Dna Binding ◽  
Target Dna ◽  
Equilibrium Shift ◽  
Dna Substrates

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Epstein–Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes

2007 ◽  
Vol 88 (7) ◽  
pp. 1876-1886 ◽  
Author(s):  
James McLaren ◽  
Martin Rowe ◽  
Paul Brennan
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Lymphoblastoid Cell Lines ◽  
Distinct Form ◽  
Post Translational Modifications ◽  
Constitutive Activation ◽  
Barr Virus ◽  
Molecular Identity ◽  
Infected Cells ◽  
Epstein Barr

Since ‘constitutive activation’ of STAT1 was first described in Epstein–Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), there has been controversy regarding the molecular identity of the STAT1 DNA-binding complex found in these cells. The post-translational modifications of STAT1 in LCLs have been analysed and an LMP1-induced STAT1 DNA-binding complex, different from that generated by alpha interferon (IFN) stimulation and not involving tyrosine phosphorylation, is demonstrated. STAT1 is serine-phosphorylated downstream of PI3K and MEK in LCLs and this modification restricts IFN-stimulated STAT1–DNA binding. These data suggest that EBV induces a distinct form of DNA-bound STAT1 in virus-infected cells.

scholarly journals Structures of GapR reveal a central channel which could accommodate B-DNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael J. Tarry ◽  
Christoph Harmel ◽  
James A. Taylor ◽  
Gregory T. Marczynski ◽  
T. Martin Schmeing
Keyword(s):  
Cell Cycle ◽  
High Resolution ◽  
Dna Binding ◽  
Central Channel ◽  
Biological Functions ◽  
Dna Mutation ◽  
Density Maps ◽  
Lysine Residues ◽  
Α Helix ◽  
Positively Charged

Abstract GapR is a nucleoid-associated protein required for the cell cycle of Caulobacter cresentus. We have determined new crystal structures of GapR to high resolution. As in a recently published structure, a GapR monomer folds into one long N-terminal α helix and two shorter α helices, and assembles into a tetrameric ring with a closed, positively charged, central channel. In contrast to the conclusions drawn from the published structures, we observe that the central channel of the tetramer presented here could freely accommodate B-DNA. Mutation of six conserved lysine residues lining the cavity and electrophoretic mobility gel shift experiments confirmed their role in DNA binding and the channel as the site of DNA binding. Although present in our crystals, DNA could not be observed in the electron density maps, suggesting that DNA binding is non-specific, which could be important for tetramer-ring translocation along the chromosome. In conjunction with previous GapR structures we propose a model for DNA binding and translocation that explains key published observations on GapR and its biological functions.

scholarly journals The Striking Flower-in-Flower Phenotype of Arabidopsis thaliana Nossen (No-0) is Caused by a Novel LEAFY Allele

Plants ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 599 ◽  
Author(s):  
Anne Mohrholz ◽  
Hequan Sun ◽  
Nina Glöckner ◽  
Sabine Hummel ◽  
Üner Kolukisaoglu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Dna Binding ◽  
Insertion Mutant ◽  
Transposon Insertion ◽  
Lateral Shoots ◽  
Target Dna ◽  
Axillary Meristems ◽  
Leafy Gene ◽  
Two Phases ◽  
Mutant Flower

The transition to reproduction is a crucial step in the life cycle of any organism. In Arabidopsis thaliana the establishment of reproductive growth can be divided into two phases: Firstly, cauline leaves with axillary meristems are formed and internode elongation begins. Secondly, lateral meristems develop into flowers with defined organs. Floral shoots are usually determinate and suppress the development of lateral shoots. Here, we describe a transposon insertion mutant in the Nossen accession with defects in floral development and growth. Most strikingly is the outgrowth of stems from the axillary bracts of the primary flower carrying secondary flowers. Therefore, we named this mutant flower-in-flower (fif). However, the transposon insertion in the annotated gene is not the cause for the fif phenotype. By means of classical and genome sequencing-based mapping, the mutation responsible for the fif phenotype was found to be in the LEAFY gene. The mutation, a G-to-A exchange in the second exon of LEAFY, creates a novel lfy allele and results in a cysteine-to-tyrosine exchange in the α1-helix of LEAFY’s DNA-binding domain. This exchange abolishes target DNA-binding, whereas subcellular localization and homomerization are not affected. To explain the strong fif phenotype against these molecular findings, several hypotheses are discussed.

scholarly journals Identification, Characterization, and Regulatory Mechanisms of a Novel EGR1 Splicing Isoform

2019 ◽  
Vol 20 (7) ◽  
pp. 1548 ◽  
Author(s):  
Vincenza Aliperti ◽  
Giulia Sgueglia ◽  
Francesco Aniello ◽  
Emilia Vitale ◽  
Laura Fucci ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Regulatory Mechanisms ◽  
Brain Diseases ◽  
Regulation Of Transcription ◽  
Biological Processes ◽  
Activation Domain ◽  
Post Translational Modifications ◽  
Pathological Conditions ◽  
Proliferation And Apoptosis

EGR1 is a transcription factor expressed in many cell types that regulates genes involved in different biological processes including growth, proliferation, and apoptosis. Dysregulation of EGR1 expression has been associated with many pathological conditions such as tumors and brain diseases. Known molecular mechanisms underlying the control of EGR1 function include regulation of transcription, mRNA and protein stability, and post-translational modifications. Here we describe the identification of a splicing isoform for the human EGR1 gene. The newly identified splicing transcript encodes a shorter protein compared to the canonical EGR1. This isoform lacks a region belonging to the N-terminal activation domain and although it is capable of entering the nucleus, it is unable to activate transcription fully relative to the canonical isoform.

scholarly journals Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications

2019 ◽  
Vol 20 (22) ◽  
pp. 5571 ◽  
Author(s):  
Claude Bobo ◽  
Claire Céré ◽  
Mélody Dufossée ◽  
Alain Dautant ◽  
Violaine Moreau ◽  
...  
Keyword(s):  
Alkaline Treatment ◽  
Regulatory Mechanisms ◽  
Electrophoretic Separation ◽  
Survival Functions ◽  
Comprehensive Description ◽  
Post Translational Modifications ◽  
Gel Composition ◽  
Spontaneous Reaction

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

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