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ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2

Winnie Tan1,2, Sylvie van Twest1, Vincent J Murphy1

  • 1Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.

Frontiers in Cell and Developmental Biology
|March 3, 2020
PubMed

Insights

ATR kinase stabilizes the Fanconi Anemia (FA) DNA repair pathway by phosphorylating FANCI, promoting FANCD2 ubiquitination and preventing its removal. This ensures efficient DNA interstrand crosslink repair for cell viability.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • DNA interstrand crosslinks (ICLs) pose a significant threat to DNA replication and cell survival.
  • The Fanconi Anemia (FA) pathway is crucial for repairing ICLs, initiated by FANCD2/FANCI ubiquitination.

Purpose of the Study:

  • To elucidate the regulatory mechanism of FANCD2/FANCI monoubiquitination by the ATR-kinase in the FA pathway.
  • To investigate how ATR-kinase activity influences the stability and function of the FANCD2/FANCI complex during DNA repair.

Main Methods:

  • Biochemical reconstitution experiments using recombinant proteins.
  • Site-directed mutagenesis to create phosphomimetic and phosphodead FANCI mutants.
  • Analysis of protein-DNA association, ubiquitination, and deubiquitination.

Main Results:

  • ATR-kinase directly phosphorylates FANCI at serine residues S556, S559, and S565.
  • Phosphorylation stabilizes the FANCI-DNA and FANCI-FANCD2 interactions, enhancing ubiquitination and inhibiting deubiquitination.
  • FANCI residues S559 and S565 are critical for protecting the complex from USP1:UAF1 deubiquitinase activity.

Conclusions:

  • ATR-kinase plays a key role in maintaining FA pathway activation by promoting FANCD2 ubiquitination.
  • ATR-mediated phosphorylation of FANCI is essential for stabilizing the active, ubiquitinated form of FANCD2 for DNA repair.
Keywords:
FANCD2FANCIdeubiquitinationphosphorylationubiquitination

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