Fok-1 Endonuclease
Fok-1 Endonuclease - Related Videos
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
0 Views •
Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
09:40
Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
0 Views •
Cited by 9 •
2011
The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
0 Views •
2020
Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.
10:59
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
0 Views •
Cited by 3 •
2021
This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.
09:53
Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
0 Views •
Cited by 2 •
2021
Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.
FAQs
Dimerization brings two catalytic domains together so they can act on opposite DNA strands. This arrangement enables formation of a double-strand break rather than cleavage of only one strand. Because productive cutting depends on the paired domains, the design and positioning of the DNA-binding components strongly influence whether a targeted break forms at the intended chromosomal location.
Cutting outside the recognition sequence separates DNA binding from the precise cleavage position. The enzyme can therefore recognize one sequence while generating a break at a defined distance from it. This Type IIS behavior provides flexibility for targeted DNA manipulation, because the binding site does not need to overlap the exact position where strand scission is required.
These DNA-binding modules provide sequence-directed targeting for the Fok-1 catalytic domain. Zinc-finger proteins and transcription activator-like effectors can be linked to engineered Fok-1 domains so that the nuclease is directed toward a chosen DNA sequence. Their targeting function complements the catalytic activity, allowing the same cleavage mechanism to be adapted for different genomic sites.
Effective cleavage depends on bringing catalytic domains into the correct relationship at the selected DNA region. A suitable arrangement allows the domains to dimerize and cut both strands, producing a defined double-strand break. If targeting does not position the domains appropriately, the desired cleavage event may not occur, limiting the usefulness of the engineered nuclease system.
A typical strategy selects a DNA-binding module for the intended sequence, connects it to an engineered Fok-1 catalytic domain, and directs the resulting system to chromosomal DNA. The paired catalytic domains then generate a targeted double-strand break. Researchers can subsequently examine the resulting gene disruption, targeted mutagenesis, precise genome editing, or DNA-repair response.
These systems are useful when an experiment requires a sequence-directed break in chromosomal DNA. Their applications include disrupting genes, introducing targeted mutations, supporting precise genome editing, and examining how cells respond to defined DNA damage. The approach is especially relevant when the research question depends on connecting a selected genomic site with a specific repair or editing outcome.
An engineered system can create a targeted double-strand break at a selected chromosomal sequence, providing a defined starting lesion for analysis. Researchers can then study the consequences of repair at that location, including outcomes associated with gene disruption, targeted mutagenesis, or precise genome editing. This links the position of DNA damage to the resulting biological change.