Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

Abstract

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by theFanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This locking of FANCI:FANCD2 complex on DNA requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2 is activated upon DNA binding and present new insights to potentially modulate monoubiquitinated FANCI:FANCD2/DNA filaments in FA cells. Fanconi Anemia | ubiquitination | DNA repair | biochemistry

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA

“…Here we show that FANCD2 ubiquitination within in the ID2 complex results in enhancement of DNA binding and a conformation that encircles DNA in a closed state. The enhancement of DNA binding and closed state observed upon ubiquitination is supported by three recent studies [33][34][35], which include cryoEM structures of human I Ub D2 Ub -DNA and chicken ID2 Ub -DNA [33,34]. Altogether, our data and these recent studies indicate that ubiquitination of FANCD2 is required to form a closed state for ID2 complex.…”

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

“…Using our system, we were able to purify ubiquitinated PCNA away from unmodified substrates via a simple affinity chromatography step. Using Avi-ubiquitin, we were able to generate native dually-monoubiquitinated FANCI:FANCD2, which has been used as a reagent to study the FA-BRCA pathway [46].…”

Formaldehyde Causes Bone Marrow Failure Linked to Transcriptional Reprogramming or Metabolic Deficiency

“…But FANCD2 focus formation is not just a marker of DNA damage; it is also necessary for replication-coupled DNA repair. To initiate repair, FANCD2 becomes clamped together with its partner protein, FANCI, and forms arrays on the DNA (Tan et al, 2020). Clamping is enforced by FANCL-mediated monoubiquitination of FANCD2.…”