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Updated: Dec 22, 2025
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Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide
Published on: June 23, 2016
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Development of a single-stranded DNA-binding protein fluorescent fusion toolbox
Katarzyna Dubiel1, Camille Henry2, Lisanne M Spenkelink3,4
1Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Nucleic Acids Research
|May 7, 2020
Summary
Engineered bacterial single-stranded DNA-binding proteins (SSBs) with internal fluorescent tags maintain function and allow visualization of DNA replication. These SSB-IDL fusions are valuable tools for studying genome maintenance.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial single-stranded DNA-binding proteins (SSBs) are crucial for DNA replication and repair by binding single-stranded DNA and recruiting other proteins.
- SSBs possess a modular structure with a DNA-binding N-terminus and an intrinsically disordered C-terminal linker (IDL) domain.
Purpose of the Study:
- To investigate the functionality of SSB-IDL fusion proteins with inserted fluorescent domains within the IDL of Escherichia coli SSB.
- To assess the utility of these fusion proteins for visualizing DNA replication and studying genome maintenance.
Main Methods:
- Construction and in vitro characterization of SSB-IDL fusion proteins with fluorescent domains inserted into the intrinsically disordered linker.
- Assessment of DNA and protein binding activities of the fusion proteins.
- Evaluation of SSB-IDL fusion protein functionality in single-molecule DNA replication assays and in vivo in Escherichia coli strains.
- Analysis of cellular fitness, SSB foci formation, and sensitivity to DNA damaging agents.
Main Results:
- SSB-IDL fusions retained DNA and protein binding activities in vitro, though cooperative DNA binding was reduced.
- Unlike C-terminal fusions, SSB-IDL fusions maintained functional protein interaction activity.
- SSB-IDL fusions were successfully visualized in single-molecule DNA replication reactions and formed detectable foci in vivo.
- Escherichia coli strains expressing SSB-IDL fusions were viable with normal growth and fitness, but showed increased sensitivity to DNA damaging agents.
Conclusions:
- SSB-IDL fusions are functional and suitable for visualizing DNA replication and genome maintenance processes.
- Internal fluorescent tagging within the SSB IDL offers advantages over C-terminal tagging for maintaining protein interaction functionality.
- These engineered SSBs provide a valuable tool for biochemical and cellular studies of DNA repair and replication mechanisms.
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