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A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest

Katharina Deiss1, Nicola Lockwood1, Michael Howell2

  • 1Protein Phosphorylation Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Nucleic Acids Research
|December 28, 2018
PubMed

Insights

The SMC5/6 complex, particularly NSE2, is crucial for the Topoisomerase 2-alpha (Topo2a)-dependent G2 arrest, a vital genome-protection pathway. This discovery offers new insights into cancer interventions for arrest-defective tumors.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The Topoisomerase 2-alpha (Topo2a)-dependent cell cycle arrest is critical for faithful sister chromatid segregation and is often dysfunctional in cancer cells.
  • Understanding this genome-protection pathway is essential for developing targeted cancer therapies, particularly exploiting synthetic lethality in tumors with defective arrest mechanisms.

Purpose of the Study:

  • To identify genes essential for the Topo2a-dependent G2 arrest using a genome-wide siRNA screen.
  • To investigate the genetic relationship between the Topo2a-dependent arrest and DNA damage checkpoints.
  • To elucidate the role of the SMC5/6 complex and NSE2 in regulating this arrest.

Main Methods:

  • Genome-wide siRNA screen using the Topo2a inhibitor ICRF193 in arrest-competent cells.
  • Counter-screening with various DNA-damaging agents.
  • Verification of findings in patient-derived fibroblasts with NSE2 mutations.
  • Imaging studies to observe protein interactions and SUMOylation.

Main Results:

  • The Topo2a-dependent G2 arrest is genetically distinct from DNA damage checkpoints.
  • Components of the SMC5/6 complex, including the E3 SUMO ligase NSE2, are essential for controlling the timing of the Topo2a-dependent G2 arrest.
  • NSE2's role was confirmed in patient fibroblasts with reduced NSE2 levels.
  • Increased interaction between Topo2a and NSE2 was observed at PML bodies during Topo2a-dependent G2 arrest.
  • Topo2a is SUMOylated by NSE2 at K1520 in an ICRF193-dependent manner, which is required for chromosome segregation but not the G2 arrest itself.

Conclusions:

  • The SMC5/6 complex, particularly NSE2-mediated SUMOylation of Topo2a at K1520, plays a critical, non-redundant role in regulating the Topo2a-dependent G2 arrest and subsequent chromosome segregation.
  • These findings highlight potential therapeutic targets for cancers with defects in this crucial genome-protection pathway.

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