Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

Martínez, David López; Kupculak, Marian; Yang, Di; Yoshikawa, Yasunaga; Liang, Chih-Chao; Wu, Ronghu; Gygi, Steven P.; Cohn, Martin A.

doi:10.1016/j.celrep.2019.05.003

Cited by 31 publications

(32 citation statements)

References 89 publications

(100 reference statements)

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“…Surprisingly, we observed that FANCI ub :FANCD2 ub forms filament-like oligomers when bound to dsDNA plasmid ( Figure 6c). Such filaments were not observed in the unmodified FANCI:FANCD2 protein preparation in the absence of presence of plasmid DNA, nor in previous investigations of human or Xenopus FANCI:FANCD2 complexes studied by EM ( Figure 6d-e and (38)(39)(40)).…”

Section: Purification Of Monoubiquitinated Fanci:fancd2 Complex Boundmentioning

confidence: 50%

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

Tan

Twest

Leis

et al. 2019

Preprint

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FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by theFanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This locking of FANCI:FANCD2 complex on DNA requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2 is activated upon DNA binding and present new insights to potentially modulate monoubiquitinated FANCI:FANCD2/DNA filaments in FA cells. Fanconi Anemia | ubiquitination | DNA repair | biochemistry

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Section: Purification Of Monoubiquitinated Fanci:fancd2 Complex Boundmentioning

confidence: 50%

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

Tan

Twest

Leis

et al. 2019

Preprint

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“…However, it's possible that other kinases could further modulate the temporal and/or spatial localization of FANCD2 and FANCI monoubiquitination in vitro or in cells. For example, Casein Kinase 2 phosphorylation of FANCD2 at a cluster of serines between residues 882-898 inhibits its DNA association and subsequent monoubiquitination (Lopez-Martinez et al, 2019), while ATM phosphorylates many residues in FANCD2 independent of monoubiquitination, but only during S-phase and after ionizing radiation (Taniguchi et al, 2002b;Ho et al, 2006). Other kinases with an important role in DNA replication and DNA damage response have yet to be explored, although several such as Chk1 and CDK also phosphorylate subunits in the FA core complex (Deans et al, 2006;Wang et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

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

Tan

Twest

Murphy

et al. 2020

Front. Cell Dev. Biol.

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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.

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“…Loss of FANCI also abolishes FANCD2 modification 11 , 12 . Monoubiquitination is a tightly controlled process, and is regulated by phosphorylation 11 , 12 , 25 – 27 . Altogether this suggests that heterodimerization of FANCD2 with FANCI is essential for this key step in the pathway.…”

Section: Introductionmentioning

confidence: 99%

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

Alcón

Shakeel

Chen

et al. 2020

Nat Struct Mol Biol

120

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Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryoEM to determine the structure of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.

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Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

Cited by 31 publications

References 89 publications

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

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

FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair

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