Fluorescent single-stranded DNA-binding proteins enable in vitro and in vivo studies

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescent single-stranded DNA-binding proteins (SSB) are crucial for understanding DNA replication and repair.
• Existing fluorescent SSB tools lack precise control over fluorophore incorporation, limiting their utility.
• A defined number of fluorophores per tetramer is needed for quantitative biochemical and biological studies.

Purpose of the Study:

• To develop fluorescent single-stranded DNA-binding proteins (SSB) with a defined number of fluorophores per tetramer.
• To enable clear in vivo visualization of SSB dynamics.
• To facilitate advanced single-molecule studies of SSB function.

Main Methods:

• Construction and purification of fluorescent SSB chimeras with varying fluorophore stoichiometry.
• Characterization of purified SSB chimeras.
• In vivo imaging experiments to visualize SSB localization and dynamics.
• Single-molecule fluorescence spectroscopy.

Main Results:

• Successfully purified fluorescent SSB chimeras with a unique, defined number of fluorescent subunits per tetramer.
• Demonstrated clear visualization of SSB in vivo using the developed chimeras.
• Showcased the utility of purified chimeras for single-molecule studies.

Conclusions:

• The developed fluorescent SSB chimeras provide precise tools for studying SSB.
• These tools enhance the ability to visualize SSB in vivo and perform quantitative single-molecule analyses.
• This work advances the understanding of DNA-binding protein mechanisms and biological roles.