Single-stranded DNA-binding Protein Displacement During Replication

Single-stranded DNA-binding proteins (SSBs) protect transiently exposed ssDNA during replication, yet DNA polymerase must somehow displace them to continue synthesis. Our latest research reveals the molecular mechanisms behind this fundamental process.

Background

During DNA replication, single-stranded DNA is temporarily exposed and vulnerable to forming secondary structures that can halt replication. SSBs bind to this exposed DNA to prevent such structures, but this creates a paradox: how does DNA polymerase access and replicate through SSB-protected regions?

Our Approach

We used a combination of cutting-edge single-molecule techniques to investigate this process:

• Single-molecule force spectroscopy to probe mechanical aspects of protein-DNA interactions
• Dual-color fluorescence imaging to visualize protein dynamics in real-time
• Molecular dynamics simulations to understand atomic-level mechanisms
• FRET measurements to detect transient protein-protein interactions

Key Discoveries

Force-Dependent SSB Function

SSB proteins have a dual role that depends on the mechanical tension in the DNA:

• At low tension: SSBs enhance replication by preventing secondary structure formation
• At high tension: SSBs become impediments that slow polymerase progression

Sequential Displacement Model

Our dual-color imaging revealed that SSBs remain stationary as DNA polymerase advances, supporting a sequential displacement mechanism rather than cooperative dissociation.

Active Destabilization by DNA Polymerase

Molecular dynamics simulations showed that DNA polymerase doesn't passively wait for SSBs to dissociate. Instead, it actively lowers the SSB dissociation energy barrier through specific interactions mediated by the SSB's C-terminal tail.

Direct Protein Interactions

FRET measurements confirmed that DNA polymerase and SSB come into close proximity during displacement events, indicating direct protein-protein interactions facilitate the handoff.

Implications

This spatiotemporal coordination between DNA polymerase and SSB represents a fundamental mechanism for resolving molecular collisions during replication. The work demonstrates that optimal replication requires SSB saturation of ssDNA, establishing a delicate balance between protection and efficiency.

Broader Impact

• Cancer research: Understanding replication machinery interactions could inform chemotherapy strategies
• Biotechnology: Engineering more efficient polymerases for PCR and sequencing applications
• Evolutionary biology: This mechanism may be conserved across all domains of life

Publication

This work is published in Nature Communications (2025) and represents a significant advance in our understanding of DNA replication dynamics at the molecular level.

Technical Innovation

The integration of multiple single-molecule techniques provided unprecedented insights into protein coordination during DNA replication, demonstrating the power of combining mechanical, optical, and computational approaches to understand complex biological processes.