The SLX4 Complex Is a SUMO E3 Ligase that Impacts on Replication Stress Outcome and Genome Stability

Abstract

The SLX4 Fanconi anemia protein is a tumor suppressor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF subunit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is mediated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO-interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA interstrand crosslink repair functions. Instead, while detrimental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is critical to prevent mitotic catastrophe following common fragile site expression.

Recruitment and positioning determine the specific role of the XPF ‐ ERCC 1 endonuclease in interstrand crosslink repair

“…We found that the XE G314E and XE ΔNSGW mutants were both able to interact normally with full‐length SLX4 (Figs 6B and EV5D). This is consistent with previous reports showing that the BTB domain is not essential for SLX4 and XPF interaction (Kim et al , 2013; Guervilly et al , 2015). One explanation for these observations is that this interaction is transient and can only be observed in the absence of the major interaction site involving the MLR domain.…”

“…Based on these observations, we suggest that the interaction site between the BTB domain of SLX4 and residues 312–315 of XPF is a minor interaction site. This is consistent with our data showing that the isolated BTB domain does not interact strongly with XPF and with results reported by Guervilly et al that show only a slight decrease in XPF binding after mutation of the SLX4 BTB domain (Guervilly et al , 2015). However, this interaction is important because the XPF ΔNSGW ‐ERCC1 mutant is deficient in ICL unhooking and repair (Figs 2 and 4).…”

SUMO conjugation – a mechanistic view

“…Interestingly, PCNA uses a similar interface to interact with Ubc9 (71). (C) Some SUMO substrates depend on a SIM close to a SCM for efficient modification (69,71,75,83,84,(113)(114)(115)(116)(117)125). (D) Posttranslational Ubc9 modifications, like sumoylation, acetylation or phosphorylation, can also regulate substrate specificity.…”

“…In contrast to the other activities that stabilize the E2 interaction with the substrate, these SIM-mediated interactions with the charged E2 might position the donor SUMO (SUMO D ) in a closed conformation, similar to that demonstrated for bona fide E3 ligases (discussed below). Intriguingly, this could explain the efficient cis-modification activities (automodifcations) of bona fide E3 ligases and diverse SIM-containing proteins like KAP1 (116) or Slx4 (117). However, detailed biochemical, and preferably structural evidence, is required to clearly distinguish between the SIM-dependent enhancement in catalysis (as it is the case for bona fide E3 ligases), as opposed to enhancement of substrate affinity.…”

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics

“…SLX4 contains SUMO-interacting motifs (SIMs) required for binding sumoylated DNA repair proteins, and SLX4 acts directly or indirectly as a SUMO E3 ligase, and its SUMO-related functions are not required for ICL repair but for a general response to replication stress. Accordingly, mutations of SLX4 SIMs do not produce ICLs hypersensitivity but cause common fragile site instability and increased mitotic catastrophe [52,53]. [65].…”