Conformational change in the herpes simplex single-strand binding protein induced by DNA
K C Dudas1, S K Scouten, W T Ruyechan
1Department of Microbiology, Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214, USA.
Biochemical and Biophysical Research Communications
|October 12, 2001
Summary
Herpes simplex virus type 1 DNA binding protein ICP8 changes its structure when it binds to single-stranded DNA, as shown by protease digestion and fluorescence experiments. This conformational change is crucial for viral DNA replication.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Herpes simplex virus type 1 (HSV-1) relies on specific viral proteins for its DNA replication cycle.
- The major single-stranded DNA binding protein, ICP8, is essential for HSV-1 DNA synthesis.
- Understanding ICP8's function requires knowledge of its structural dynamics during DNA binding.
Purpose of the Study:
- To investigate the conformational changes of HSV-1 ICP8 protein upon binding to single-stranded DNA.
- To identify regions of ICP8 protected from protease digestion when bound to DNA.
- To assess alterations in ICP8's biophysical properties indicative of structural shifts.
Main Methods:
- Protease digestion assays were performed on ICP8 in the presence and absence of single-stranded DNA oligonucleotides.
- ICP8 was chemically modified with fluorescein-5-maleimide (FM) to enable fluorescence-based measurements.
- Antibody quenching and fluorescence polarization techniques were used to analyze ICP8's conformation.
Main Results:
- Protease digestion revealed distinct cleavage patterns, with protection of amino acids 293-806 in the presence of DNA.
- Fluorescein-labeled ICP8 showed increased susceptibility to antibody quenching and decreased fluorescence polarization when bound to DNA.
- These biophysical changes indicate a significant structural rearrangement of ICP8 upon single-stranded DNA interaction.
Conclusions:
- ICP8 undergoes a substantial conformational change when it binds to single-stranded DNA.
- The DNA binding induces a more compact or altered structure, shielding specific regions from proteolysis.
- These findings provide critical insights into the mechanism of HSV-1 DNA replication mediated by ICP8.
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