Integron integrase binds to bulged hairpin DNA

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Gene cassettes are mobile DNA elements within integrons.
• Site-specific recombination mediated by tyrosine recombinases facilitates their translocation.
• The attC site is crucial for integrase binding and recombination, but its sequence variability suggests structural determinants are key.

Purpose of the Study:

• To investigate the DNA binding mechanism of integrase to the attC site.
• To identify specific sequence and structural features of attC that influence integrase binding.
• To elucidate the role of the catalytic residue in covalent complex formation.

Main Methods:

• Electrophoresis mobility shift assay (EMSA) was used to examine integrase binding to modified attC oligonucleotides.
• Analysis of DNA binding and covalent complex formation upon mutation of the catalytic tyrosine residue (Y312).

Main Results:

• Integrase binding strength and strand choice are significantly influenced by inner repeats, a central triplet, and single-nucleotide asymmetries within the attC site.
• The recombinase binds to a bulged hairpin structure in attC, with hairpin distortion potentially dictating site orientation.
• A covalent DNA-protein linkage is formed, confirmed by complex stability under denaturing conditions and abolished upon Y312 mutation, though DNA binding persists.

Conclusions:

• Integrase recognition of attC sites relies on structural features, particularly a bulged hairpin, rather than solely sequence.
• The catalytic tyrosine residue is essential for forming the covalent linkage, but not for initial DNA binding.
• Understanding these interactions provides insights into the mechanism of gene cassette recombination in integrons.