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Single-Molecule Dynamics at a Bacterial Replication Fork after Nutritional Downshift or Chemically Induced Block in

Rogelio Hernández-Tamayo1,2, Hannah Schmitz3,2, Peter L Graumann1,2

  • 1SYNMIKRO, LOEWE Center for Synthetic Microbiology, Marburg, Germany rogelio.hernandez@synmikro.uni-marburg.de peter.graumann@synmikro.uni-marburg.de.

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Summary

Bacterial replication forks show remarkable plasticity, adjusting protein dynamics to various stresses like nutrient changes or DNA damage. This adaptability ensures replication can quickly resume after obstacles are removed.

Keywords:
Bacillus subtilisDNA helicaseDNA polymeraseDNA primaseDNA replicationhelicasereplicationsingle-molecule microscopysingle-molecule trackingstringent response

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • DNA replication is essential for cell survival and must adapt to environmental changes.
  • Replication forks, the sites of DNA synthesis, are complex multiprotein machines.
  • Bacterial cells face various stresses, including nutrient depletion (stringent response), DNA damage, and polymerase inhibition, which can impede replication.

Purpose of the Study:

  • To investigate the single-molecule dynamics of key replication proteins (helicase DnaC, primase DnaG, polymerase DnaE) in *Bacillus subtilis* under different stress conditions.
  • To understand how replication forks respond to transient blocks caused by DNA damage, polymerase inhibition, or nutritional downshift.
  • To elucidate the mechanisms of protein recruitment and association with replication forks during stress.

Main Methods:

  • Single-molecule tracking of DnaC, DnaG, and DnaE in live *Bacillus subtilis* cells.
  • Real-time observation of protein dynamics during various induced stress conditions.
  • Development of a novel tool to quantify exchange rates between protein diffusion and static binding.

Main Results:

  • Replication fork proteins exhibit differential responses to distinct stress conditions.
  • DNA primase (DnaG) binding is reduced during stringent response, DNA damage, or nutritional downshift, suggesting (p)ppGpp binding prevents its recruitment.
  • Turnover rates of DnaC, DnaG, and DnaE increase during nutritional downshift-induced replication blocks, indicating fork plasticity.
  • Replication forks remain intact under all tested stress conditions, facilitating rapid restart.

Conclusions:

  • Bacterial replication forks display significant plasticity, dynamically adjusting protein interactions to overcome diverse obstacles.
  • The stringent response primarily affects primase recruitment rather than displacing already bound primase.
  • Independent recruitment of DnaG and DnaE suggests distinct regulatory mechanisms at the replication fork.
  • The observed plasticity ensures replication continuity and rapid recovery following stress events.