Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
Nucleic Acids Res. 2012 May;40(10):4553-61. doi: 10.1093/nar/gks053. Epub 2012 Jan 28.
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.
FANCI 和 FANCD2 形成复合物，在范可尼贫血（FA）途径中对链间 DNA 交联（ICLs）的修复起关键作用。FANCD2 被 FA 核心复合物单泛素化，该复合物由 10 种 FA 蛋白组成，包括作为催化 E3 亚基的 FANCL。FANCD2 的单泛素化可以在体外使用纯化的最小成分如 FANCI、E1、UBE2T（E2）和 FANCL（E3）重新构成；然而，与 FANCD2 的体内单泛素化相比，其效率相当低。在这项研究中，我们发现各种形式的 DNA，如单链、双链和分支 DNA，在体外强烈刺激 FANCD2 的单泛素化，达到与体内单泛素化相当的水平。这种 FANCD2 单泛素化的刺激严格需要 FANCI，表明 FANCD2 单泛素化可能发生在 FANCI-FANCD2 复合物中。一种在 DNA 结合中缺陷的 FANCI 突变体在体外的 FANCD2 单泛素化中也明显缺陷。在存在 5' 翻折 DNA 的情况下，一种模拟停滞复制叉的 DNA 底物，约 70%的输入 FANCD2 被单泛素化，而在没有 DNA 的情况下，不到 1%的 FANCD2 被单泛素化。因此，DNA 可能是 FANCD2 正确单泛素化所需的未识别因素。
