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Mapping the functional domains of bacteriophage lambda integrase protein
Y W Han1, R I Gumport, J F Gardner
1Department of Microbiology, University of Illinois at Urbana-Champaign 61801.
Journal of Molecular Biology
|January 21, 1994
Summary
Bacteriophage lambda integrase (Int) mutants were genetically isolated and characterized to understand site-specific recombination. Key regions of Int were identified, including those involved in DNA binding, catalysis, and protein-protein interactions.
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- Bacteriophage lambda utilizes a site-specific recombination system for integration and excision from the host chromosome.
- The phage-encoded integrase (Int) protein, a 356-amino acid enzyme, possesses type I topoisomerase activity essential for DNA strand exchange.
- Int features distinct DNA-binding domains that recognize specific arm-type and core-type DNA sequences.
Purpose of the Study:
- To elucidate the mechanism of bacteriophage lambda site-specific recombination.
- To identify functional domains within the integrase (Int) protein through a genetic approach.
- To isolate and characterize Int mutants defective in excisive recombination and DNA binding.
Main Methods:
- Development of a genetic screen to isolate Int mutants deficient in excisive recombination in vivo.
- Utilized bacteriophage P22 challenge-phage assays to screen mutants for binding to P'123 arm-type sites.
- Characterization of 78 isolated mutants, including mutational mapping, sequencing, DNA-binding assays, in vitro negative dominance, topoisomerase activity assays, and resolution of recombination intermediates.
Main Results:
- Identified a region of 88 amino acids in the middle of Int potentially involved in Int-Int interactions.
- Localized a catalytic site region around Arg212 responsible for type I topoisomerase activity.
- Determined that residues near the carboxyl terminus enhance Int binding to arm-type sites, possibly via interaction with the N-terminal DNA-binding region.
- Found that residues at the C-terminus may modulate the cleavage and religation activities of Int.
Conclusions:
- The study provides critical insights into the functional organization of bacteriophage lambda integrase.
- Specific regions of Int are implicated in DNA binding, protein-protein interactions, catalysis, and regulation of recombination.
- This genetic analysis advances the understanding of site-specific recombination mechanisms in bacteriophages.