Characterization of bacteriophage lambda excisionase mutants defective in DNA binding.

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage lambda excisionase (Xis) is essential for site-specific DNA recombination.
• Xis protein binds cooperatively to specific DNA sites and interacts with host factors and integrase for efficient excision.
• The secondary structure of Xis is predicted to contain an amphipathic helix (residues 18-28).

Purpose of the Study:

• To investigate the role of the putative amphipathic helix and other regions in the DNA binding and functional activity of bacteriophage lambda Xis protein.
• To identify the specific amino acid residues and structural features responsible for Xis DNA binding and recombination activity.

Main Methods:

• Site-directed mutagenesis was used to substitute alanines for polar amino acids within the predicted amphipathic helix of the Xis protein.
• Mutant Xis proteins were analyzed for their ability to promote excisive recombination in vivo.
• DNA binding assays were performed to assess the effect of mutations on Xis-DNA interactions.

Main Results:

• Mutant Xis proteins with alanine substitutions in the amino-terminal region of the amphipathic helix showed reduced in vivo excision activity and impaired DNA binding.
• An alanine substitution at glutamic acid 40 also resulted in altered DNA binding.
• These findings suggest that the hydrophilic face of the alpha-helix and the region around glutamic acid 40 are critical for Xis DNA binding.

Conclusions:

• The hydrophilic face of the N-terminal amphipathic alpha-helix and the region including glutamic acid 40 are important DNA-binding surfaces of the bacteriophage lambda Xis protein.
• These structural elements are crucial for the protein's ability to mediate excisive recombination.