The molecular basis of co-operative DNA binding between lambda integrase and excisionase

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Nucleoprotein complexes regulate protein-DNA interactions essential for biological processes.
• Bacteriophage Lambda Integrase (Int) mediates site-specific DNA recombination, forming intasomes.
• Excisionase (Xis) protein modulates Int activity, promoting excision while inhibiting integration.

Purpose of the Study:

• Investigate the functional role of a specific Int mutation (E47K) previously linked to excision defects.
• Elucidate the molecular mechanism by which Int and Xis interact to regulate recombination.
• Determine the structural and functional requirements for co-operative DNA binding mediated by Int.

Main Methods:

• Site-directed mutagenesis of the bacteriophage Lambda Integrase gene.
• Biochemical assays to assess protein-protein and protein-DNA interactions.
• Analysis of recombination efficiency for integration and excision pathways.

Main Results:

• The int 2268 ts mutant harbors an E47K substitution in the Int protein.
• This E47K substitution significantly destabilizes the interaction between Int and Xis, causing an excision-specific defect.
• Negatively charged residues at Int position 47 are essential for co-operative DNA binding with Xis and Int self-association.
• Substitutions at Int 47 impact both integration and excision efficiencies by affecting DNA binding.

Conclusions:

• The Int E47 residue plays a critical role in mediating distinct co-operative binding interactions with Xis and other Int proteins.
• A single surface on the Int amino-terminal domain is involved in both Int-Xis and Int-Int co-operative binding.
• Understanding these interactions provides insights into the regulation of bacteriophage DNA recombination.