Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• The Fanconi anaemia pathway is crucial for repairing DNA interstrand crosslinks.
• Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central step in this pathway.
• FANCD2 ubiquitination occurs before FANCI ubiquitination, but FANCD2 is also deubiquitinated faster, potentially leading to a FANCI-ubiquitinated ID2 complex (I_Ub D2).

Purpose of the Study:

• To elucidate the structural basis of the I_Ub D2 complex bound to DNA.
• To understand the role of FANCI ubiquitination in maintaining FANCD2 ubiquitination and DNA repair.
• To investigate the deubiquitination dynamics of the ID2 complex.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of the I_Ub D2-DNA complex at 4.1 Å resolution.
• Structural analysis of the ID2 complex in different ubiquitination states (ID2_Ub, I_Ub D2, I_Ub D2_Ub) bound to DNA.
• Biochemical assays to assess deubiquitination resistance and susceptibility.

Main Results:

• The I_Ub D2-DNA complex adopts a closed conformation, clamping onto DNA, similar to other ID2 ubiquitination states.
• The structure reveals exposed target lysines (FANCD2 K561 and FANCI K523), priming them for ubiquitination.
• The I_Ub D2-DNA complex is resistant to deubiquitination, while ID2_Ub-DNA is efficiently deubiquitinated by USP1-UAF1 unless FANCI is also ubiquitinated.
• FANCI ubiquitination stabilizes FANCD2 ubiquitination by preventing excessive deubiquitination and enabling re-ubiquitination.

Conclusions:

• FANCI ubiquitination plays a critical role in stabilizing the FANCI-FANCD2 complex on DNA.
• This stabilization is essential for maintaining FANCD2 ubiquitination, thereby ensuring efficient DNA interstrand crosslink repair.
• The findings provide structural insights into the regulatory mechanisms of the Fanconi anaemia pathway.