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Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study
Tihomir Solomun1, Leo Cordsmeier1,2, Dorothea C Hallier1,3,4
1Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin 12205, Germany.
The Journal of Physical Chemistry. B
|September 13, 2023
Summary
Gene-V protein (G5P) binds single-stranded DNA (ssDNA), unzipping hairpin structures. This protein-DNA interaction, studied via fluorescence, reveals G5P
Area of Science:
- Molecular Biology
- Biophysics
- Structural Biology
Background:
- Gene-V protein (G5P/GVP) is a single-stranded DNA-binding protein (SBP) essential for bacteriophage f1 DNA synthesis and repair.
- G5P forms a left-handed helical protein-DNA filament by binding two antiparallel ssDNA strands cooperatively.
Purpose of the Study:
- To investigate the interaction of G5P with different DNA structures using fluorescence studies.
- To elucidate the effect of G5P on DNA secondary structures like hairpins.
Main Methods:
- Fluorescence spectroscopy of end-labeled DNA oligonucleotides (Cy3-fluorophore and BHQ2-quencher).
- Fluorescence microfluidic mixing experiments.
- Electrophoretic mobility shift assay (EMSA).
Main Results:
- G5P binding to unstructured ssDNA resulted in near-complete fluorescence quenching, indicating precise DNA end alignment.
- G5P induced the unzipping of DNA hairpin stems, confirmed by fluorescence and EMSA.
- Significant protein-induced fluorescence enhancement (PIFE) of Cy3 was observed.
Conclusions:
- G5P actively disrupts ssDNA secondary structures, consistent with other SBPs.
- The binding mechanism involves precise alignment and structural modification of DNA.
- Fluorescence techniques effectively probe G5P-DNA interactions and structural changes.
