Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA

Nucleic Acids Res. 2014 May;42(9):5657-70. doi: 10.1093/nar/gku198. Epub 2014 Mar 12.

Abstract

FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • DNA, Circular / chemistry
  • DNA, Viral / chemistry*
  • Fanconi Anemia Complementation Group D2 Protein / chemistry*
  • Fanconi Anemia Complementation Group L Protein / chemistry
  • Fanconi Anemia Complementation Group Proteins / chemistry*
  • Fanconi Anemia Complementation Group Proteins / genetics
  • Humans
  • Inhibitor of Differentiation Protein 2 / chemistry
  • Nucleosomes / chemistry
  • Plasmids / chemistry
  • Protein Binding
  • Sf9 Cells
  • Spodoptera
  • Substrate Specificity
  • Ubiquitination*

Substances

  • DNA, Circular
  • DNA, Viral
  • FANCD2 protein, human
  • FANCI protein, human
  • Fanconi Anemia Complementation Group D2 Protein
  • Fanconi Anemia Complementation Group Proteins
  • ID2 protein, human
  • Inhibitor of Differentiation Protein 2
  • Nucleosomes
  • FANCL protein, human
  • Fanconi Anemia Complementation Group L Protein