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Top1 and Top2 promote replication fork arrest at a programmed pause site.

Mélanie V Larcher1, Philippe Pasero1

  • 1Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier 34396, France.

Genes & Development
|January 4, 2020
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Summary

Programmed DNA replication pausing involves interactions between fork protection complexes and topoisomerases. This study reveals Topoisomerase I physically interacts with the Tof1-Csm3 complex, regulating fork pausing in yeast.

Keywords:
Csm3Mrc1RFBTof1Top1Top2replication fork pausingreplisometopoisomerase

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Programmed fork pausing is a crucial cellular process that arrests DNA replication forks at specific sites.
  • Replication fork barriers are not passive roadblocks but involve dynamic interactions with fork machinery.

Purpose of the Study:

  • To investigate the molecular mechanisms underlying programmed replication fork pausing.
  • To determine the role of DNA topoisomerases in regulating fork pausing at the ribosomal DNA replication fork barrier (RFB) in budding yeast.

Main Methods:

  • Co-immunoprecipitation assays to detect physical interactions between Tof1-Csm3 and Topoisomerase I.
  • Analysis of replication fork pausing in yeast mutants lacking Topoisomerase I or with mutations in the Tof1-Top1 interaction domain.
  • Functional assays to assess the impact of topoisomerase activity on RFB function.

Main Results:

  • The fork protection complex Tof1-Csm3 physically interacts with DNA topoisomerase I (Top1) at replication forks via the C-terminal domain of Tof1.
  • Impaired fork pausing at the rDNA RFB was observed in the absence of Top1 or in a tof1 mutant unable to bind Top1.
  • Topoisomerase II (Top2) can partially compensate for the loss of Top1 function in regulating fork pausing.

Conclusions:

  • Topoisomerases play a previously unrecognized role in the regulation of programmed replication fork pausing.
  • The interaction between Tof1-Csm3 and Top1 is critical for efficient fork pausing at the rDNA RFB in Saccharomyces cerevisiae.
  • These findings highlight a novel regulatory mechanism for DNA replication fork control.