The role of FANCI phosphorylation in Fanconi anemia pathway regulation

Krejčová Kateřina, Bouřa Evžen, Šilhán Jan

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 542/2, Praha 6, 16000,

katerina.krejcova@uochb.cas.cz

Fanconi anemia is an autosomal recessive disorder caused by mutation in one of Fanconi genes and it is manifested by developmental abnormalities, bone marrow failure, predisposition to cancer, cellular sensitivity to cross-linking agents and many other symptoms [1]. Proteins encoded by Fanconi genes and some other proteins are part of Fanconi anemia pathway (FA pathway), which is responsible for  DNA repair of an interstrand cross-link (ICL)[2]. The repair by this pathway requires monoubiquitylation of FANCD2, which is induced and regulated by ATR dependent FANCI phosphorylation. The FANCI phosphorylation initiates the FA pathway but the molecular mechanism of this initialization is not known. Furthermore the proper function of entire pathway requires both: sequence of phosphorylation events of FANCI and monoubiquitylation of FANCD2:FANCI complex [3].

In this project we study molecular mechanisms of initiation and regulation of FA pathway by FANCI phosphorylation. Therefore we have created phosphomimetic mutants of FANCI to investigate their role in processes leading to FANCD2 monoubiquitylation. Our aim is to reveal how the phosphorylation of FANCI affects DNA binding and also DNA binding of the FANCI:FANCD2 complex. Since both DNA and FANCI phosphorylation are required for proper FANCD2 monoubiquitylation, we measured and compared these individual phosphorylation mimetic and phosphorylation dead mutants of FANCI in DNA binding assays. In ongoing experiments we are testing the role of FANCI phosphorylation in FANCD2 binding and in monoubiquitylation assays. Although FANCI dramatically improved the binding of its partner FANCD2 to DNA, mutations or phosphomimetic had only marginal effect on the DNA binding. Therefore, we need to investigate further on how exactly the phosphorylation of FANCI triggers the FANCD2 monoubiquitylation and regulates entire FA pathway progression.

1.         Auerbach, A. D. & Allen, R. G. Leukemia and preleukemia in Fanconi anemia patients. A review of the literature and report of the International Fanconi Anemia Registry. Cancer Genet. Cytogenet. 51, 1–12 (1991).

2.         Sato, K., Toda, K., Ishiai, M., Takata, M. & Kurumizaka, H. DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. Nucleic Acids Research 40, 4553–4561 (2012).

3.         Ishiai, M. et al. FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nature Structural & Molecular Biology 15, 1138–1146 (2008).

The project was supported by the Czech Science Foundation (17-21649Y). We thank to Academy of Sciences Czech Republic for support in form of J.E. Purkyne Fellowship awarded to J.S. The project was also supported by the Academy of Sciences Czech Republic (RVO: 61388963).