Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Herpes simplex virus type 1 (HSV-1) DNA replication initiates at a specific origin, oriS.
• The origin binding protein (OBP) is essential for viral DNA replication.
• oriS contains recognition sites for OBP arranged in palindromic structures.

Purpose of the Study:

• To investigate the specific conformation of oriS bound by OBP.
• To determine the role of DNA secondary structures in OBP binding and oriS function.
• To elucidate the mechanism of OBP-mediated initiation of HSV-1 DNA replication.

Main Methods:

• Site-directed mutagenesis to destabilize and restore hairpin structures in oriS.
• Electrophoretic mobility shift assays (EMSAs) to assess OBP binding to oriS variants.
• In vitro DNA replication assays to evaluate the functional impact of mutations.
• ATP hydrolysis assays to measure OBP enzymatic activity.

Main Results:

• OBP binds to a specific box III-box I hairpin structure within the oriS* conformation.
• Mutations destabilizing this hairpin abolish OBP binding and inhibit DNA replication.
• Restoration of the hairpin through compensatory mutations rescues OBP binding and replication activity.
• The oriS* hairpin structure efficiently activates OBP's ATP hydrolysis.

Conclusions:

• The box III-box I hairpin in oriS* is essential for OBP recognition and initiation of HSV-1 DNA replication.
• OBP exhibits high specificity for distinct oriS conformations during replication initiation.
• A two-step model for oriS-mediated replication initiation involving OBP binding to duplex and hairpin DNA is proposed.
• Conserved palindromic sequences in alpha herpesvirus origins suggest a conserved mechanism.