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Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization
A J Doherty1, S R Ashford, H S Subramanya
1Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
The Journal of Biological Chemistry
|May 10, 1996
Summary
Researchers purified and characterized bacteriophage T7 DNA ligase, a monomeric enzyme that ligates various DNA fragments. This DNA ligase was successfully crystallized, marking a first for this enzyme family.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- DNA ligases are essential enzymes for DNA replication, repair, and recombination.
- Bacteriophage T7 DNA ligase is a well-studied enzyme with potential applications in molecular biology.
- Crystallization of DNA ligases provides insights into their structure-function relationships.
Purpose of the Study:
- To overexpress, purify, and characterize bacteriophage T7 DNA ligase.
- To investigate the DNA ligation activity and inhibition of the enzyme.
- To crystallize the enzyme for structural analysis.
Main Methods:
- Polymerase chain reaction (PCR) amplification and cloning of the T7 DNA ligase gene.
- Bacterial expression and protein purification.
- Biochemical assays for DNA ligase activity and inhibition studies.
- X-ray crystallography for structure determination.
Main Results:
- High-level expression and homogeneous purification of T7 DNA ligase (60-70 mg/L).
- The enzyme functions as a monomer and ligates nicked, cohesive, and blunt-ended DNA fragments.
- Enzyme activity is inhibited by a nonhydrolyzable ATP analogue.
- Crystals of T7 DNA ligase were obtained, belonging to the P2(1)2(1)2 space group, diffracting to 2.6 Å.
Conclusions:
- Bacteriophage T7 DNA ligase is a monomeric enzyme with broad substrate specificity for DNA ligation.
- The successful crystallization of T7 DNA ligase provides a foundation for future structural studies.
- This represents the first crystal structure of a DNA ligase enzyme, offering new avenues for understanding DNA ligation mechanisms.