Novel, fluorescent, SSB protein chimeras with broad utility

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The Escherichia coli single-stranded DNA binding protein (SSB) is crucial for DNA metabolism, organizing genome maintenance and stabilizing single-stranded DNA (ssDNA).
• Existing methods for producing functional fluorescent SSB are often cumbersome, hindering real-time studies.

Purpose of the Study:

• To develop a novel expression system for producing chimeric SSB proteins that are easily purified and functional.
• To utilize these chimeric SSBs for advanced biochemical and in vivo studies of DNA processing and protein interactions.

Main Methods:

• A dual plasmid expression system was engineered to create chimeric SSB proteins composed of histidine-tagged and fluorescent protein (FP)-fused subunits.
• Purified chimeric SSBs were used in single-molecule studies and biochemical assays without further modification.
• In vivo interaction studies were performed using the developed chimeric proteins.

Main Results:

• The expression system yielded milligram quantities of easily purified chimeric SSB proteins.
• Chimeric SSBs demonstrated full functionality comparable to wild-type SSB in all assays, unaffected by FP fusions.
• Single-molecule studies provided new insights into the RecBCD mechanism.
• The study provided the first in vivo evidence of an interaction between RecG and SSB.

Conclusions:

• The novel chimeric SSB proteins are a significant advancement, simplifying the production and use of functional fluorescent SSB for various biological studies.
• These tools facilitate in vivo, in vitro, and single-molecule investigations of DNA metabolism and protein interactions.
• The findings open new avenues for exploring genome maintenance mechanisms and protein collaborations within the cell.