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

Winnie Tan; Sylvie van Twest; Vincent J Murphy; Andrew J Deans

doi:10.3389/fcell.2020.00002

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

Front Cell Dev Biol. 2020 Feb 4:8:2. doi: 10.3389/fcell.2020.00002. eCollection 2020.

Authors

Winnie Tan^{1

2}, Sylvie van Twest¹, Vincent J Murphy¹, Andrew J Deans^{1

2}

Affiliations

¹ Genome Stability Unit, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
² Department of Medicine (St Vincent's Hospital), The University of Melbourne, Melbourne, VIC, Australia.

Abstract

DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair.

Keywords: FANCD2; FANCI; deubiquitination; phosphorylation; ubiquitination.