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Characterization of bacteriophage lambda excisionase mutants defective in DNA binding.

E H Cho¹, R Alcaraz, R I Gumport

¹Department of Science Education, Chosun University, Kwangju, Korea.

Journal of Bacteriology

|September 27, 2000

Summary

Bacteriophage lambda excisionase (Xis) protein is crucial for DNA recombination. Mutations in its predicted alpha-helix and at glutamic acid 40 impair DNA binding and excision, suggesting these regions are key DNA interaction sites.

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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.

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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.