The Ubiquitination of UFL1 by the APC/C complex may affect the DNA repair pathway choice

DNA double-strand breaks (DSBs) are the most toxic DNA lesions contributing to genome instability. DSBs can be mainly repaired by homologous recombination (HR) and non- homologous end-joining (NHEJ). By exploring the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, we identified the cell division cycle 23 protein as a new substrate, with Ser588 being its putative phosphorylation site, upon DNA damage. CDC23 is subunit of the Anaphase-promoting complex/cyclosome (APC/C), a multimeric E3 ubiquitin ligase complex involved in DNA repair. We observed by mutating the CDC23 in the Serine target of CDK9-55 phosphorylation, a defect in the repair pathway choice observing an impairment in HR. Through LC/MS analysis we identified the UFM1 specific ligase 1 (UFL1) as a new target of the APC/C-CDC23(S588A) during DNA damage. Recently, UFL1, the only known E3-like ligase that mediates the UFMylation process, was demonstrated to be involved in HR pathway as part of ATM activation. We demonstrated how the poly-ubiquitination of UFL1, mediated by the APC/C-CDC23 complex upon DSBs, does not lead to its proteasomal degradation but probably involved in its cellular localization change. The recognition of target substrates by the APC/C complex depends on the specificity of two co-activators, Cell Division Cycle 20 (CDC20) and Fizzy and Cell Division Cycle 20 Related 1 (FZR1 or CDH1), which bind degron motifs in the substrate’s sequences, named ABBA-BOX and D-BOX, respectively. We investigated the presence of these recognition motifs and identified both putative ABBA-BOX and D-BOX domains in UFL1 protein sequence. Our preliminary data describe a possible new regulation of the repair pathway choice between HR and NHEJ upon DSB formation, suggesting the potential role of the APC/C in selecting the appropriate repair pathway possibly by regulating UFL1 protein polyubiquitination. Moreover, these results represent further evidence of the key role of the UFMylation process in the activation of Homologous recombination. These findings pave the way for new biomarkers as well as new potential therapeutic approaches in cancer treatment.