Related Experiment Videos
The catalytic domain of lambda site-specific recombinase
R S Tirumalai1, E Healey, A Landy
1Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
Summary
The study identifies a minimal catalytic domain (C170) of the phage lambda integrase protein, crucial for DNA recombination. This fragment aids in understanding the chemistry of DNA cleavage and ligation in site-specific recombinases.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Phage lambda integrase (Int) is a site-specific recombinase essential for viral DNA integration.
- Int utilizes a covalent phosphotyrosine intermediate for recombination at att sites.
Purpose of the Study:
- To identify and characterize the minimal catalytically competent domain of phage lambda integrase.
- To elucidate the functional and structural domains of Int involved in DNA recombination.
Main Methods:
- Cloning and characterization of carboxy-terminal fragments of Int (C65 and C170).
- Assays for topoisomerase activity and DNA binding (gel shift).
- Analysis of protease sensitivity and structural data (crystal structure).
Main Results:
- A 188-amino acid fragment (C170) was identified as the minimal catalytic domain.
- C170 exhibits topoisomerase activity and forms covalent phosphotyrosine complexes.
- C170 shows reduced binding to att site DNA, suggesting domain specialization.
- The active site Tyr-342 is located in a flexible, protease-sensitive loop within C170.
Conclusions:
- Phage lambda Int comprises three functional domains: DNA recognition (N-terminal), core-site binding/protein interactions (middle), and catalysis (C-terminal).
- The C170 fragment represents a conserved Int family domain, facilitating study of DNA cleavage and ligation chemistry.
- Understanding Int domains advances knowledge of site-specific recombination mechanisms.