Regulation of catalysis by the smallpox virus topoisomerase

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Poxvirus type IB topoisomerases are essential enzymes for DNA replication and transcription.
• These enzymes function by cleaving and rejoining DNA strands through a covalent phosphotyrosine intermediate.
• Understanding topoisomerase structure-function relationships is crucial for antiviral drug development.

Purpose of the Study:

• To investigate the functional importance of specific amino acid residues in poxvirus topoisomerase activity.
• To elucidate the role of the active site in DNA cleavage, religation, and product release.
• To identify potential targets for smallpox antiviral therapies.

Main Methods:

• Site-directed mutagenesis was used to create twenty-two amino acid substitutions in the poxvirus topoisomerase.
• In vitro assays were performed to measure DNA relaxation, single-cycle cleavage, and equilibrium cleavage-religation.
• High-level expression of mutant enzymes in Escherichia coli was assessed for cellular effects.

Main Results:

• Alanine substitutions at 14 positions impaired topoisomerase function, highlighting critical protein-DNA interface contacts.
• Mutations D168A and E124A unexpectedly accelerated the DNA cleavage rate.
• High-level expression of the D168A mutant inhibited bacterial growth, suggesting a role in cellular processes.

Conclusions:

• Specific amino acid side chains are critical for poxvirus topoisomerase DNA binding and catalytic activity.
• Asp(168) acts as a key regulator of the active site, balancing enzymatic steps.
• These findings provide a basis for designing novel topoisomerase poisons as smallpox antivirals.