Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability

Abstract

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC. Author summary DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that can compromise genomic instability, thereby driving carcinogenesis. DNA repair by homologous recombination (HR) is critical in faithfully repairing this type of DNA damage and the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have emerged as critical regulators of this pathway. Still, it remains largely unclear how they promote HR-mediate DNA repair. Here, we mapped their respective proximal interactome using the BioID approach and we identified the spliceosome machinery as an integral component of our proximal mapping. Importantly, several members of the spliceosome machinery have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome.