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Characterization of the herpes simplex virus origin binding protein interaction with OriS
D J Hazuda1, H C Perry, A M Naylor
1Department of Virus and Cell Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
The Journal of Biological Chemistry
|December 25, 1991
Summary
Herpes simplex virus origin binding protein (OBP) specifically binds a 10-base pair sequence in OriS for viral DNA replication. High-affinity binding requires specific DNA contexts and involves interactions on one face of the DNA helix.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Herpes simplex virus origin binding protein (OBP) is crucial for viral DNA replication.
- OBP specifically recognizes and binds to sequences within viral replication origins.
Purpose of the Study:
- To investigate the precise DNA sequence and structural requirements for OBP binding to the HSV origin, OriS.
- To characterize the nature of OBP-DNA interactions at the molecular level.
Main Methods:
- Expression of OBP (full-length and fusion proteins) in Escherichia coli.
- Analysis of OBP binding to variant DNA sequences and heteroduplexes.
- Determination of binding affinity (Ka) and mutational analysis of the OBP-binding site.
Main Results:
- The OBP-binding site within OriS is a 10-base pair sequence (5' CGTTCGCACT 3').
- High-affinity binding (Ka ≈ 0.3 nM) requires at least 15 base pairs and is sensitive to mutations in the central CGC sequence.
- OBP binds with high affinity to DNA heteroduplexes, indicating specific interactions localized to one face of the DNA helix within the major groove.
Conclusions:
- OBP recognizes a specific DNA sequence through base-mediated interactions primarily on one face of the helix.
- The spatial orientation of OBP-binding sites within OriS is critical for its function in viral DNA replication.