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

Rose Westhorpe1, Johann J Roske1, Joseph T P Yeeles1

  • 1Protein and Nucleic Acid Chemistry Division, Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

Molecular Cell
|September 5, 2024
PubMed

Insights

Replication forks encountering Topoisomerase 1 cleavage complexes (Top1-ccs) are surprisingly stable. Fork stability depends on the DNA template, fork protection proteins, and converging forks, revealing key replisome remodeling mechanisms.

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.
Keywords:
CMG helicaseTop1Top1 cleavage complexfork protection complexreplication stressreplisometopoisomerase

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