Interstrand crosslinks (ICLs) are cytotoxic form of DNA damage. ICLs precipitate Fanconi anaemia (FA) a rare genetic disease with patients carrying biallelic mutations in one of some 22 genes (FANC genes). The patients suffer from developmental defects, small stature, bone marrow failure and often succumb to cancer. Cell lines derived from FA patients are hypersensitive to DNA crosslinking agents, such as Cisplatin. Leading to implication of FANC genes in ICL repair and further postulation of FA DNA repair pathway[1]. The hallmark of FA dependent ICL repair is FANCD2 monoubiquitylation, a crucial reaction required for further ICL excisions by DNA nuclease complex SLX4-XPF-ERCC1 [2-4]. Additinally, genetic studies have shown that phosphorylation of FANCI triggers FANCD2 monoubiquitylation. In this work we investigate the role of phosphorylated FANCI in the process of initiation and progression of ICL repair.
To dissect the molecular mechanisms FANCI phosphorylation, we generated mutants of five known ATR phosphorylation. These sites were mutated to Asp as a phosphorylation mimic and Ala as a control. To test the influence of FANCI phosphorylation we used various DNA substrates mimicking DNA replication including single stranded and double stranded DNA. We have measured fluorescent anisotropy, gel based DNA binding assays and kinetics of FANCD2 monoubiquitylation reaction. We have validated DNA binding of FANCD2 and FANCI in comparison with FANCD2-FANCI complex. Here FANCD2 bound to DNA with lower affinity then FANCI. Although we did not observe striking differences between individual DNA structures, combining the two proteins led to significant increase of DNA binding affinity. This is in accordance with the fact that FANCD2 is monoubiquitylated on DNA. Although the presence of monoubiquitylated FANCD2 has not revealed significant changes in DNA binding affinities we have observed significant stimulatory effect of DNA binding using phosphomimetic mutants of FANCI. Our data bring the evidence that ATR phosphorylation triggers the FANCI and FANCD2 ubiquitylation by its localisation on DNA.
The work was supported by the Czech Science Foundation (17-21649Y). The project was also supported by the Academy of Sciences Czech Republic (RVO: 61388963).