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Replication forks reverse at high frequency upon replication stress in Physarum polycephalum.

Chrystelle Maric1, Marianne Bénard

  • 1Institut Jacques Monod, UMR 7592 CNRS-Université Paris Diderot-Paris7, 15 Rue Hélène Brion, 75205, Paris CEDEX 13, France, maric@ijm.univ-paris-diderot.fr.

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Summary

Hydroxyurea treatment reveals that reversed replication forks and joint DNA molecules form during DNA replication stress. These structures, including chicken foot structures, are transient and detectable due to slowed replication.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA replication is a complex process susceptible to various stresses.
  • Replication fork dynamics and the structures formed under stress are crucial for genome stability.
  • Understanding these mechanisms is key to comprehending DNA repair and disease development.

Purpose of the Study:

  • To investigate the formation and dynamics of DNA replication structures under stress.
  • To elucidate the role of hydroxyurea in revealing replication-dependent joint DNA molecules and chicken foot structures.
  • To determine the frequency and dependency of reversed replication forks during replication stress.

Main Methods:

  • Synchronous cell culture of Physarum polycephalum.
  • Treatment with hydroxyurea to induce replication stress.
  • Detection and analysis of replication-dependent joint DNA molecules and chicken foot structures.
  • Microscopic observation of replication fork progression and regression.

Main Results:

  • Hydroxyurea addition post-S phase initiation allowed detection of joint DNA molecules and chicken foot structures.
  • A high frequency of reversed replication forks was observed under replication stress.
  • Formation of reversed forks depended on joint DNA molecules, hydroxyurea, and origin reinitiation.
  • Chicken foot structures arise from both regressed joint DNA molecules and stalled forks.

Conclusions:

  • Hydroxyurea treatment enables the sequential detection of replication intermediates under stress.
  • Replication fork regression is a transient event, made detectable by hydroxyurea-induced slowing of fork progression.
  • The study provides insights into the mechanisms of replication fork plasticity and genome maintenance.