The response to DNA damage and DNA replication stress is orchestrated by the ATM and ATR kinases that direct the phosphorylation of key proteins that carry out this stress response. Knowledge of their substrates is key to both identifying and elucidating DNA repair responses. In this study we examine one such recently identified substrate of these kinases (Matsuoka et al, submitted), the KIAA1794 protein, which we discovered to be encoded by the FANCI gene.
FANCI is a FANCD2 paralog required for crosslink repair
FANCI depletion caused a phenotype consistent with a role in DNA crosslink repair. FANCI is highly conserved from humans to Dictyostelium, but absent in budding and fission yeast. The interior of FANCI, like that of FANCD2 is composed of ARM repeats which fold into superhelical helices, suggesting that these proteins have an extended structure. A crucial finding was a short stretch of similarity to Sea Urchin FANCD2, a central component of the Fanconi anemia pathway. This conserved region contained a key lysine known to be monoubiquitinated in FANCD2 that is critical for FANCD2 function. FANCI contains a weak but significant similarity throughout the length of the human FANCD2 protein. As FANCI and FANCD2 are approximately the same size, show similarity along their length, and both have an extended region of super helical ARM repeats, it is likely that these proteins were derived from a common ancestral gene duplication event that subsequently diverged through evolution to produce two functionally distinct paralogs.
FANCI is mutated in FA-I cells
As KIAA1794/FANCI showed characteristics of a Fanconi anemia protein we anticipated that previously unassigned Fanconi patients might harbor mutations in our gene. We were able to obtain BD0952 cells from the FA-I complementation group. FA-I cells are MMC-sensitive and fail to monoubquitinate FANCD2 or form FANCD2 foci in response to DNA damage. Retroviral expressed FANCI complemented FA-I mutants for all three phenotypes. DNA sequence analysis revealed two amino acid changes in the FANCI protein. Complementation analysis reveals that the R1285Q mutation is responsible for the defect in BD0952 cells and fails to restore MMC-resistance and FANCD2 regulation. The R1285Q mutation is present in both copies of FANCI in these cells consistent with family consanguinity. The complementation, coupled with the identification of inactivating mutations in BD0952 cells, unambiguously identifies KIAA1794 as the FANCI gene. FANCI in the three other FA-I cell lines (Levitus et al., 2004
) remains to be examined for the presence of mutations to confirm the cell-fusion data that placed them in the same complementation group as BD0952 cells.
FANCI is monoubiquitinated
Like FANCD2, FANCI has a slower migrating form that several lines of evidence suggest is due to monoubiquitination. First, the key lysine responsible for FANCD2 ubiquitination is conserved in FANCI. Secondly, FANCA mutant cells defective for the FA ubiquitin ligase complex fail to generate the slower migrating form. Third, mutation of this lysine, K523R, in FANCI prevents formation of the slower migrating form. Finally, FANCI antibodies specifically immunoprecipitate epitope tagged ubiquitin covalently attached to FANCI. Like FANCD2, the ubiquitinated form of FANCI is enriched on chromatin and the K523R FANCI mutant does not get loaded onto chromatin
The significance of FANCI monoubiquitination is illustrated by the fact that the K523R mutant of FANCI does not itself become monoubiquitinated and does not form damage foci. When introduced into U2OS cells, which express a WT FANCI allele, this mutant reduced FANCD2 monoubiquitination and foci formation. Furthermore, the K523R mutant fails to fully complement FANCD2 ubiquitination or MMC sensitivity of BD0952 cells indicating that ubiquitination is important for FANCI function. As this line is not a null mutant, it is possible that this intermediate FANCD2 monoubiquitination and MMC-resistance phenotype might represent interallelic complementation.
FANCI and D2 form an interdependent ID complex that is required for ubiquitination and chromatin association
We anticipated that FANCI would associate with FANCD2 because FANCI protein formed foci that colocalized with FANCD2, depletion of FANCI significantly reduced the monoubiquitination of FANCD2 preventing its inclusion into DNA damage induced foci, and FA-I cells have completely lost their ability to monoubiquinate FANCD2 despite fully formed FA complex (Levitus et al., 2004
). Indeed, FANCI forms a complex with FANCD2, with approximately 20% of FANCD2 coimmunoprecipitating with tagged FANCI protein. The FANCI-FANCD2 complex, which we propose to call the Fanconi anemia ID complex, thus joins the Fanconi anemia core complex as a new downstream complex necessary for ICL repair and homologous recombination.
Just as the FANCI protein monoubiquitination is required for FANCD2 ubiquitination and foci formation, FANCD2 and FANCD2 ubiquitination is required for FANCI ubiquitination. Thus, these two paralogs are interdependent and employ a dual ubiquitination mechanism to affect downstream effector function. Furthermore, phosphorylation of FANCD2 is required for its own efficient ubiquitination and therefore for the efficient ubiquitination of FANCI. By extension, FANCI phosphorylation is also expected to be an essential event for the FA pathway following DNA damage and during normal S phase progression. This phosphorylation-ubiquitination cascade culminating in chromatin loading of the ID complex offers an exquisite control at sites of stalled forks. The ID complex, when correctly placed, can direct repair pathways to remove the crosslinks and repair the DNA so that replication can resume and cells can survive. Without this key event, cells are prone to genomic instability that can lead both to increased cell death, stem cell depletion, and tumorigenesis.
Among the key questions that remain to be answered is how the ID complex is recognized for ubiquitination. Is it directly ubiquitinated by the FANCL ligase complex or is there a more complex relationship among different ligases converging on this pathway. What is the function of ID ubiquitination and why must both components be monoubiquitinated? Is it merely required for chromatin loading or does it direct repair in the same way that PCNA ubiquitination directs repair? Does it represent a ubiquitin code that directs specific repair pathways? The identification of FANCI and the Fanconi anemia ID complex should now allow these issues to be approached with greater clarity.