The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DOT Integrase (IntDOT) is a tyrosine recombinase enzyme essential for the integration and excision of the CTnDOT integrative and conjugative element (ICE).
• DNA integration involves two strand exchanges between specific att sites, forming a Holliday Junction (HJ) intermediate.
• Previous studies identified Valine 95 (V95) as critical for HJ resolution by IntDOT.

Purpose of the Study:

• To identify additional IntDOT residues involved in Holliday Junction resolution.
• To elucidate the role of hydrophobic interactions in IntDOT function and complex formation.

Main Methods:

• Site-directed mutagenesis was used to substitute key residues in the IntDOT N-terminal helix.
• Electrophoretic mobility shift assays (EMSAs) were employed to analyze IntDOT-DNA complex formation.

Main Results:

• Two additional hydrophobic residues, Alanine 92 (A92) and Phenylalanine 99 (F99), were identified as important for IntDOT-mediated HJ resolution.
• Mutations at A92 and F99 resulted in the formation of aberrant IntDOT-DNA complexes, as observed in EMSAs.
• These findings suggest a collective role for V95, A92, and F99 in IntDOT function.

Conclusions:

• The hydrophobic residues V95, A92, and F99 in the IntDOT N-terminal helix are critical for resolving DNA Holliday Junctions.
• These residues likely mediate hydrophobic interactions essential for forming higher-order IntDOT complexes and facilitating DNA integration/excision reactions.
• This study provides new insights into the molecular mechanisms of tyrosine recombinase function.