DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins.

Area of Science:

• Molecular Biology
• Biophysics
• Genomics

Background:

• Single-stranded DNA-binding proteins (SSBs) are crucial for protecting exposed single-stranded DNA (ssDNA) during replication.
• The mechanism by which DNA polymerases (DNAp) displace SSBs to facilitate replication is not fully understood.

Purpose of the Study:

• To investigate the molecular mechanisms of SSB displacement by DNAp during replication.
• To elucidate the spatiotemporal coordination between DNAp and SSB.

Main Methods:

• Single-molecule force spectroscopy
• Dual-color imaging
• Molecular dynamics simulations
• Förster Resonance Energy Transfer (FRET)

Main Results:

• T7 SSB's effect on replication is force-dependent, enhancing it at low tension and impeding it at high tension.
• SSBs remain stationary as DNAp advances, supporting a sequential displacement model.
• DNAp actively reduces the SSB dissociation energy barrier via interactions with the SSB C-terminal tail.

Conclusions:

• The spatiotemporal coordination between DNAp and SSB is vital for resolving molecular collisions during replication.
• This coordination ensures both DNA replication processivity and genomic integrity.
• A balance between SSB protection and replication efficiency is critical for optimal DNA synthesis.