DNA stimulates FANCD2 monoubiquitination in the human FANCI-FANCD2 complex

Assoc Rsrch Scientist Mol Biophys & Biochem
333 Cedar St, New Haven, CT 06510-3206

         FANCD2 and 15 other proteins whose deficiency leads to Fanconi anemia (FA) function in a common network to repair crosslinked DNA. DNA damage stimulates monoubiquitination of FANCD2 and FANCI  (the ID complex). In vivo, monoubiquitination of both proteins requires the FA core complex, a multisubunit ubiquitin ligase comprised of FANCA, -B, -C, -E, -F, -G, -L and -M plus cofactors.  
         We previously described a minimal in vitro system for monoubiquitination of human FANCI by the FANCL E3 ligase and the E2 partner, UBE2T (1). Interestingly, FANCI and FANCD2 ubiquitination was minimal when purified human ID complex was used as a substrate in this system. These results agree with the recent crystal structure of an ID complex wherein the target lysines in FANCD2 and FANCI are buried in the FANCD2-FANCI interface, inaccessible for ubiquitination (2).
         FANCI, FANCD2 and the ID complex are DNA binding proteins (1-4). Importantly, addition of duplex and branched DNA, but not unstructured single-stranded DNA, to the in vitro ID complex ubiquitination reaction greatly stimulated FANCD2 monoubiquitination, whereas FANCI ubiquitination was relatively unchanged. Mutations that disrupt the DNA binding ability of the ID complex impaired FANCD2 ubiquitination. These results, and recent similar results using homologous chicken proteins (5), suggest that the ID complex undergoes a conformational change upon DNA binding that unblocks the lysine residue targeted for monoubiquitination in FANCD2.

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