Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant

Abstract

FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2−/− DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.

Germline Mutations in Cancer Predisposition Genes are Frequent in Sporadic Sarcomas

“…In particular, Asn580 is located in a region concentrated with polar residues shown to interface with FANCD2. While the specific effect of these mutations remains to be functionally confirmed, the potential deleterious consequence on the activity of FANCD2 in addition to the functional studies reported in literature demonstrating the loss of protein function in the FANC -family genes 32 , 33 collectively provide some evidence favouring the assignment of pathogenicity to the missense mutations we observed in this study. Importantly, the consistency of our findings with a larger, more powered study such as the ISKS indicates that our bioinformatics approach can reasonably discover potentially pathogenic germline mutations in our cohort.…”

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

“…The control beads were prepared by the same method, in the absence of RAD51. For the cell-based pull-down assay, HeLa cell extracts were prepared with lysis buffer, containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM NaF, 2 mM Na 3 VO 4 and 1x protease inhibitor cocktail (Nacalai Tesque), as previously described (54). DT40 cell extracts were prepared by the same method used for the HeLa cell extract preparation.…”

“…Gel-filtration analyses were performed with 100 μl of FANCI (5 μM), FANCD2 (5 μM) or the I-D complex (5 μM), as described previously (54,55). The peak fractions at the 7.5-15 ml elution volume were analyzed by 8% SDS-PAGE with Coomassie Brilliant Blue staining.…”

Potential Strategies to Target Protein–Protein Interactions in the DNA Damage Response and Repair Pathways

“…These conflicting observations hamper mechanistic justification for therapeutic PPI inhibition of the ID complex at this stage. However, a point mutant of chicken FANCD2 (L234R that corresponds to the L231R mutation identified in a human FA patient) is defective in monoubiquitination and FANCI PPI, and DT40 cells expressing this mutant are >10-fold more sensitive to cisplatin than are those expressing WT, indicating functional justification for targeting this PPI. Structure of the ID complex includes the huge interface of the PPI .…”