Related Experiment Video
Updated: May 25, 2026

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
Published on: March 14, 2019
DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI
Koichi Sato1, Kazue Toda, Masamichi Ishiai
1Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
Abstract:
FANCI and FANCD2 form a complex, and play essential roles in the repair of interstrand DNA crosslinks (ICLs) by the Fanconi anemia (FA) pathway. FANCD2 is monoubiquitylated by the FA core complex, composed of 10 FA proteins including FANCL as the catalytic E3 subunit. FANCD2 monoubiquitylation can be reconstituted with purified minimal components, such as FANCI, E1, UBE2T (E2) and FANCL (E3) in vitro; however, its efficiency is quite low as compared to the in vivo monoubiquitylation of FANCD2. In this study, we found that various forms of DNA, such as single-stranded, double-stranded and branched DNA, robustly stimulated the FANCD2 monoubiquitylation in vitro up to a level comparable to its in vivo monoubiquitylation. This stimulation of the FANCD2 monoubiquitylation strictly required FANCI, suggesting that FANCD2 monoubiquitylation may occur in the FANCI-FANCD2 complex. A FANCI mutant that was defective in DNA binding was also significantly defective in FANCD2 monoubiquitylation in vitro. In the presence of 5' flapped DNA, a DNA substrate mimicking the arrested replication fork, about 70% of the input FANCD2 was monoubiquitylated, while less than 1% FANCD2 monoubiquitylation was observed in the absence of the DNA. Therefore, DNA may be the unidentified factor required for proper FANCD2 monoubiquitylation.
More Related Videos
08:56In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay
Published on: May 5, 2020
14:34Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay
Published on: December 25, 2021
Related Concept Videos
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Protein Complexes with Interchangeable Parts
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Anaphase Promoting Complex
Regulation of the Unfolded Protein Response
Rab Cascades