Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function...

Area of Science:

• Molecular Biology
• Enzymology
• Structural Biology

Background:

• Bse634I restriction endonuclease is a tetrameric type IIF enzyme requiring two recognition sites for optimal activity.
• Tetrameric enzymes often exhibit complex regulatory mechanisms influencing their catalytic efficiency.
• Understanding enzyme quaternary structure is crucial for elucidating DNA-protein interactions and enzymatic function.

Purpose of the Study:

• To investigate the role of tetramerization in the catalytic activity and regulation of Bse634I restriction endonuclease.
• To determine the effect of disrupting the tetramerization interface on enzyme structure and function.
• To explore the mechanism of autoinhibition and its relief in type IIF restriction enzymes.

Main Methods:

• Site-directed mutagenesis was employed to disrupt the tetramerization interface of Bse634I, creating a dimeric mutant (W228A).
• Enzyme activity assays were performed using plasmid DNA with single and double recognition sites.
• Cleavage analysis of immobilized oligonucleotides was used to study synaptic complex formation.

Main Results:

• Disruption of the tetramerization interface converted the tetrameric Bse634I into a dimer (W228A mutant).
• The dimeric mutant exhibited enhanced catalytic efficiency, cleaving single- and double-site DNA comparably to the tetramer on double-site DNA.
• Bse634I forms catalytically competent synaptic complexes by bridging multiple DNA molecules, relieving autoinhibition.

Conclusions:

• The tetrameric structure of Bse634I imposes autoinhibition on single-site DNA cleavage, mediated by inter-dimer cross-talk.
• A single W228A mutation converts the type IIF tetramer into an active dimeric enzyme, akin to type IIP endonucleases.
• While enhancing activity, the dimeric mutant shows reduced stability against chemical, thermal, and proteolytic challenges.