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SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination

Jinhua Han1, Ting Liu1, Michael S Y Huen2

  • 1Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.

The Journal of Cell Biology
|June 25, 2014
PubMed

Insights

Researchers discovered SIVA1 is crucial for targeting the RAD18 enzyme to PCNA, a key step in DNA damage tolerance. This finding clarifies how DNA repair mechanisms function following cellular damage.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Translesion DNA synthesis (TLS) is a vital DNA damage tolerance mechanism conserved across species.
  • Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulatory event in TLS.
  • The RAD6-RAD18 complex is known to monoubiquitinate PCNA, but the targeting mechanism was unclear.

Purpose of the Study:

  • To elucidate the mechanism by which the RAD6-RAD18 complex is targeted to PCNA.
  • To identify novel regulators of PCNA monoubiquitination.

Main Methods:

  • Affinity purification to isolate PCNA-containing complexes.
  • Co-immunoprecipitation assays to study protein interactions.
  • siRNA-mediated knockdown to assess protein function in vivo.
  • UV sensitivity and mutation assays to evaluate DNA repair capacity.

Main Results:

  • SIVA1 was identified as a protein that constitutively interacts with PCNA through a conserved motif.
  • SIVA1 knockdown impaired RAD18-dependent PCNA monoubiquitination and Polη focus formation.
  • SIVA1 acts as a molecular bridge, connecting RAD18 to PCNA and facilitating PCNA monoubiquitination.
  • SIVA1 deficiency resulted in increased UV sensitivity and mutation rates.

Conclusions:

  • SIVA1 is essential for targeting the RAD18 E3 ligase to its substrate PCNA.
  • This interaction is critical for efficient PCNA monoubiquitination and subsequent DNA repair.
  • The study reveals SIVA1 as a key accessory protein required for RAD18 ligase activity on PCNA.

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