title: Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Simonne
authors.initials: S
authors.lastname: Longerich
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Youngho
authors.initials: Y
authors.lastname: Kwon
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Miaw-Sheue
authors.initials: MS
authors.lastname: Tsai
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Aye Su
authors.initials: AS
authors.lastname: Hlaing
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Gary M
authors.initials: GM
authors.lastname: Kupfer
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA patrick.sung@yale.edu.
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004270:DNA, Circular / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000737:chemistry*; D052240:Fanconi Anemia Complementation Group L Protein / Q000737:chemistry; D051856:Fanconi Anemia Complementation Group Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2 / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D054875:Ubiquitination
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; C499905:FANCD2 protein, human; C519921:FANCI protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D009707:Nucleosomes; C499908:FANCL protein, human; D052240:Fanconi Anemia Complementation Group L Protein