Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage lambda excision from the E. coli chromosome requires Int and Xis proteins.
• Understanding the precise roles of Int and Xis in recombination is crucial for deciphering viral integration and excision mechanisms.

Purpose of the Study:

• To purify and characterize the lambda Xis protein.
• To investigate the in vitro activity of Xis in lambda DNA recombination.
• To elucidate the functional relationship between Xis and Int proteins.

Main Methods:

• Complementation assay using purified Int and E. coli host factor extract.
• In vitro recombination assays with plasmid DNA containing attL and attR sites.
• Polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
• Assays for endonuclease, topoisomerase, and DNA binding activities.

Main Results:

• Lambda Xis protein was purified and demonstrated to be essential for in vitro excision.
• Xis (8.8 kDa) lacks endonuclease, topoisomerase, and direct DNA binding activities.
• Xis stimulates excisive recombination while inhibiting integrative recombination.
• Xis protects Int protein from heat inactivation, indicating a potential protein-protein interaction.

Conclusions:

• The lambda Xis protein is a key regulator of bacteriophage lambda DNA recombination.
• Xis functions by modulating the activity of Int, promoting excision over integration.
• Xis likely interacts directly with Int, influencing its stability and DNA binding properties.