Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining

Abstract

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.

RAD54L2 counters TOP2-DNA adducts to promote genome stability

“…Collectively, the IP-MS experiments suggested an interplay between sumoylation and the ability of RAD54L2 to bind TOP2α, TOP2β, and ZNF451. In line with our data, recent studies have identified RAD54L2 as a strong SUMO interactor and predicted 12 potential SIMs in RAD54L2 ( 23 – 25 ).…”

Paradoxes of Cellular SUMOylation Regulation: A Role of Biomolecular Condensates?

“…The apparent loss of interaction with polySUMO2 caused by mutation of pSIM33 is likely due to the loss of structural integrity of the head domain as mutation of pSIM33 also abolished XRCC4's ability to bind XLF, a known binding partner for the head domain. On the other hand, mutation of the novel non-canonical SIM KDVSF at position 102-106 to alanines did not have noticeable effects on the overall structural integrity of the mutated proteins and abrogated binding of XRCC4 to polySUMO2 (Cabello-Lobato et al, 2022). In addition to the short SUMO-interacting motif discussed above, the ZZ Zinc finger domain of HERC2 has also been shown to bind stoichiometrically with SUMO1 with an affinity of ∼3 µM and SUMO2 with a lower affinity, around 60 µM (Danielsen et al, 2012).…”

“…). Similar to conventional SIMs, this new motif has a hydrophobic patch, but an unprecedented positively charged lysine residue at the core (Cabello-Lobato et al, 2022). This motif is found in the N-terminal head domain of X-ray repair cross-complementing 4 (XRCC4), a DNA double-strand break repair protein that preferentially interacts with polySUMO2 over monomeric SUMO (Cabello-Lobato et al, 2022).…”

“…Similar to conventional SIMs, this new motif has a hydrophobic patch, but an unprecedented positively charged lysine residue at the core (Cabello-Lobato et al, 2022). This motif is found in the N-terminal head domain of X-ray repair cross-complementing 4 (XRCC4), a DNA double-strand break repair protein that preferentially interacts with polySUMO2 over monomeric SUMO (Cabello-Lobato et al, 2022). A putative SIM with a canonical sequence of VITL at position 33-36 (pSIM33) in XRCC4 was previously identified, and its mutation led to the disruption of the binding of polySUMO2/3 (González-Prieto et al, 2021).…”

SUMO and the DNA damage response

“…Two further SBDs have been identified: the MYM zinc finger [ 16 ] and the ZZ domain (Type III interactors) [ 17 ]. Furthermore, yet uncharacterised, SBDs can be inferred from SUMO interactions not involving these domains/motifs [ 18 ]. SUMOylation and ubiquitination co-operate in DNA damage repair (DDR) signalling [ 19 , 20 ].…”