Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Topoisomerase 1 cleavage complexes (Top1-ccs) are DNA-protein crosslinks that impede DNA replication.
• Top1-ccs inhibitors are crucial for inducing replication stress in research and clinical applications.
• The precise response of the replication machinery (replisome) to Top1-ccs collisions is not well understood.

Purpose of the Study:

• To elucidate the mechanisms by which the replisome responds to collisions with Top1-ccs.
• To investigate the factors influencing replication fork stability upon encountering Top1-ccs.

Main Methods:

• Reconstitution of budding yeast replisomes using purified proteins.
• In vitro assembly of site-specific Top1-ccs.
• Analysis of replication fork stalling and collapse dynamics.
• Nascent-strand DNA mapping.
• Cryoelectron microscopy (cryo-EM) of stalled replication forks.

Main Results:

• Replication forks colliding with Top1-ccs exhibit unexpected stability.
• Fork stability is modulated by the DNA template strand involved, the Tof1-Csm3 (TIMELESS-TIPIN) complex, and replication fork convergence.
• Replisome remodeling is identified as a critical initial response to Top1-ccs.

Conclusions:

• The study reveals novel mechanisms of replication fork stabilization and remodeling in response to Top1-ccs.
• Findings enhance understanding of how DNA replication machinery navigates and responds to Top1-induced DNA damage.
• These insights are vital for optimizing the use of Top1 inhibitors in cancer therapy and fundamental research.