Attenuating functions of the C terminus of lambda integrase

Area of Science:

• Molecular Biology
• Enzymology
• Structural Biology

Background:

• Tyrosine recombinases, unlike monomeric topoisomerases, utilize multi-protein complexes for DNA synapsis and strand exchange.
• Lambda integrase (Int) is a key enzyme in site-specific recombination, requiring coordinated actions within a complex.

Purpose of the Study:

• To investigate the functional consequences of mutations in the catalytic domain of lambda integrase (Int).
• To understand how specific mutations affect recombination and topoisomerase activity.
• To elucidate the structural basis for regulated DNA cleavage and strand exchange in Int.

Main Methods:

• Analysis of three lambda integrase (Int) catalytic domain mutants: A241V, I353M, and W350ter.
• Assays for recombination and topoisomerase activity using truncated substrates and Holliday junctions.
• Structural modeling to predict the impact of mutations on protein conformation and residue positioning.

Main Results:

• Mutant Int enzymes exhibit defective recombination but enhanced topoisomerase activity.
• Mutants display increased DNA cleavage on isolated att sites and can perform individual strand exchanges.
• Structural modeling suggests mutations destabilize the C-terminal beta-strand (beta7), affecting catalytic residue positioning (Y342, K235).

Conclusions:

• Anchoring of beta7 normally restrains catalytic residues, attenuating cleavage activity in most contexts.
• Release of beta7 in recombination complexes allows active conformations for coordinated strand exchange.
• Mutations disrupting beta7 packing lead to unregulated activity, favoring DNA cleavage over efficient recombination.