Related Experiment Video
Updated: Jul 19, 2026
13:30
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Temperature-sensitive mutants of the EcoRI endonuclease
R S Muir1, H Flores, N D Zinder
1Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Journal of Molecular Biology
|February 7, 1998
Summary
Researchers created temperature-sensitive EcoRI endonuclease mutants to study DNA-protein interactions. These mutations reveal critical residues affecting enzyme structure and function, offering insights into molecular mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- EcoRI endonuclease is a key tool in recombinant DNA technology.
- It serves as a model for understanding sequence-specific DNA-protein interactions.
- Understanding EcoRI's structure-function relationship is crucial for its applications.
Purpose of the Study:
- To isolate and characterize temperature-sensitive (TS) mutants of EcoRI endonuclease.
- To investigate the impact of specific mutations on enzyme stability, structure, and activity.
- To gain insights into the critical residues and structural elements governing EcoRI function.
Main Methods:
- Isolation and characterization of 11 TS EcoRI endonuclease mutants.
- In vivo and in vitro activity assays at different temperatures (30°C and 42°C).
- Analysis of enzyme expression, solubility, and DNA cleavage activity.
- Structural analysis using X-ray crystallography of the wild-type EcoRI-DNA complex.
Main Results:
- The majority of isolated mutants exhibited temperature sensitivity for in vivo function but were stable in vitro.
- Mutations were found to cluster in specific regions of the enzyme, with some affecting inter-residue interactions and local structures.
- Two mutations (G78D, A235E) potentially impact the dimer interface, and one (P90S) is near the active site.
- Mutants were classified into two groups based on in vivo restriction and in vitro activity, suggesting differences in kinetics or mechanism.
Conclusions:
- The identified mutations provide valuable insights into EcoRI endonuclease structure and function.
- These TS mutants highlight the importance of specific residues for enzyme folding and activity at elevated temperatures.
- The findings complement existing genetic, biochemical, and structural data on EcoRI, aiding in the understanding of DNA-protein interactions.
Related Concept Videos
01:16In vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:11Restriction Enzymes
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...
01:20Mismatch Repair
Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
01:27Diversity of Archaea III
Crenarchaeota, a prominent phylum of Archaea, is remarkable for its ability to thrive in extreme environments characterized by high temperatures and acidity. These microorganisms inhabit sulfuric hot springs, volcanic systems, and submarine hydrothermal vents, where temperatures often exceed 100°C. The unique adaptations of Crenarchaeota not only allow survival under such extreme conditions but also provide insights into the mechanisms of life in primordial Earth-like environments.Morphological...
01:29Diversity of Archaea IV
Hyperthermophilic archaea are a group of extremophiles thriving at temperatures above 80°C, often in hydrothermal vents and volcanic soils where conditions surpass the boiling point of water. At such temperatures, proteins, membranes, and DNA in most organisms degrade, but hyperthermophiles have evolved remarkable adaptations to maintain stability and function.Unique Cellular FeaturesHyperthermophilic membranes are composed of a monolayer of biphytanyl tetraether lipids, which resist thermal...