Replication dynamics of recombination-dependent replication forks

Area of Science:

• Molecular Biology
• Genetics
• DNA Replication

Background:

• Replication forks restarted by homologous recombination (HR) are known to be error-prone.
• Understanding the polymerase dynamics during HR restart is crucial for comprehending genome stability.

Purpose of the Study:

• To visualize and characterize the in vivo replication dynamics of HR-restarted forks at a specific replication barrier (RTS1) in S. pombe.
• To elucidate the roles of different DNA polymerases (Pol δ, Pol ε, Pol α) in synthesizing DNA during HR restart.
• To investigate the progression of HR-restarted forks through replication fork barriers and the impact of lagging strand resection.

Main Methods:

• Polymerase usage sequencing to visualize in vivo replication dynamics.
• Monte Carlo simulations to model replication processes.
• Genetic manipulation (deletion of pku70) to alter lagging strand resection during HR restart.

Main Results:

• HR-restarted forks synthesize both DNA strands using Pol δ for extended distances (up to 30 kb) without switching to a Pol δ/Pol ε configuration.
• DNA Polymerase α (Pol α) is not significantly utilized on either strand during HR restart, suggesting a gap-filling mechanism by Pol δ on the lagging strand.
• HR-restarted forks can progress through replication fork barriers that arrest canonical replication forks.
• Altering lagging strand resection impacts HR restart, but the leading strand initiation site remains stable, indicating the robustness of the 3' single strand.

Conclusions:

• HR-restarted forks exhibit a distinct polymerase usage pattern, primarily relying on Pol δ for extensive synthesis on both leading and lagging strands.
• These forks are capable of navigating challenging DNA structures like fork barriers, contributing to genome integrity.
• Lagging strand resection plays a role in HR restart, but the fundamental process of leading strand initiation remains stable, highlighting the resilience of the replication machinery.