Related Experiment Video
Updated: Apr 21, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
7.9K
Massively parallel determination and modeling of endonuclease substrate specificity
Summer B Thyme1, Yifan Song2, T J Brunette2
1Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA sthyme@gmail.com.
Nucleic Acids Research
|November 13, 2014
Summary
Researchers identified novel homing endonucleases by mining genome databases and screening bacterial systems. Deep sequencing characterized their DNA cleavage, revealing specificities that computational models partially predicted, offering evolutionary insights.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- Homing endonucleases are powerful tools for genome engineering.
- Understanding their specificity is crucial for precise DNA targeting.
- Novel endonuclease discovery can expand genome editing capabilities.
Purpose of the Study:
- To identify and characterize novel homing endonucleases.
- To investigate the substrate specificity and kinetics of newly discovered endonucleases.
- To explore the evolutionary mechanisms behind endonuclease specificity.
Main Methods:
- Genome database mining for putative endonuclease target sites.
- High-throughput activity screening in a bacterial selection system.
- Deep sequencing to monitor DNA cleavage and characterize endonuclease kinetics.
- 3D structure-based computational modeling to predict endonuclease specificity.
Main Results:
- Identification and characterization of several novel homing endonucleases.
- Detailed analysis of substrate specificity and cleavage kinetics using deep sequencing.
- Computational models partially recapitulated experimental endonuclease specificities.
- Insights into the generation of alternative endonuclease specificities during evolution.
Conclusions:
- Novel homing endonucleases were successfully identified and characterized.
- Deep sequencing and computational modeling are effective tools for endonuclease analysis.
- Understanding endonuclease evolution provides a basis for designing new genome editing tools.
More Related Videos
Related Concept Videos
01:13Induced-fit Model
92.3K
Most chemical reactions in cells require enzymes—biological catalysts that speed up the reaction without being consumed or permanently changed. They reduce the activation energy needed to convert the reactants into products. Enzymes are proteins, that usually work by binding to a substrate—a reactant molecule that they act upon.
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
92.3K
01:11Restriction Enzymes
40.3K
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
40.3K