Distinct functional roles for the SLX4 ubiquitin-binding UBZ domains mutated in Fanconi anemia

Abstract

Defects in SLX4, a scaffold for DNA repair nucleases, result in Fanconi anemia (FA), due to the defective repair of inter-strand DNA crosslinks (ICLs). Some FA patients have an SLX4 deletion removing two tandem UBZ4-type ubiquitin-binding domains that are implicated in protein recruitment to sites of DNA damage. Here, we show that human SLX4 is recruited to sites of ICL induction but that the UBZ-deleted form of SLX4 in cells from FA patients is not. SLX4 recruitment does not require either the ubiquitylation of FANCD2 or the E3 ligases RNF8, RAD18 and BRCA1. We show that the first (UBZ-1) but not the second UBZ domain of SLX4 binds to ubiquitin polymers, with a preference for K63-linked chains. Furthermore, UBZ-1 is required for SLX4 recruitment to ICL sites and for efficient ICL repair in murine fibroblasts. The SLX4 UBZ-2 domain does not bind to ubiquitin in vitro or contribute to ICL repair, but it is required for the resolution of Holliday junctions in vivo. These data shed light on SLX4 recruitment, and they point to the existence of currently unidentified ubiquitylated ligands and E3 ligases that are crucial for ICL repair.

Noncovalent Interactions with SUMO and Ubiquitin Orchestrate Distinct Functions of the SLX4 Complex in Genome Maintenance

“…Consistent with previous reports (Kim et al, 2013; Lachaud et al, 2014; Yamamoto et al, 2011), SLX4 WT but not SLX4 2ubz suppressed the sensitivity of SLX4 -null cells to MMC (Fig. 3A).…”

“…How SLX4 recognizes different types of DNA lesions is just beginning to unfold. It has been suggested that SLX4 uses its ubiquitin-binding zinc finger (UBZ) domains to engage the mono-ubiquitylated FANCD2 or other poly-ubiquitylated proteins at ICLs (Lachaud et al, 2014; Yamamoto et al, 2011). Furthermore, a fraction of SLX4 is recruited to telomeres via its interaction with TRF2 (Wan et al, 2013; Wilson et al, 2013).…”

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…154 While Yamamoto and colleagues established that the FANCP/SLX4 UBZ domain and the monoubiquitination of FANCD2 are required for FANCP/SLX4 targeting to nuclear foci, a recent report has contradicted these findings and shown that SLX4 recruitment occurs independent of FANCD2 monoubiquitination. 154,161 Taken together, these findings suggest that one major function for chromatin-bound monoubiquitinated FANCD2 may be the recruitment of structure-specific endonucleases that mediate key incision steps during the process of ICL repair.…”

Defects in homologous recombination repair behind the human diseases: FA and HBOC

“…It has been reported that SLX4 is recruited by monoubiquitinated FANCD2 (Yamamoto et al 2011). However, there is a conflicting report (Lachaud et al 2014), and the UBZ domain is generally considered to bind to K63-linked polyubiquitin (Lachaud et al 2014). How SLX4 is tethered to the sites of damage and how the D2-I complex affects unhooking are important issues that need to be resolved.…”