Nucleolar accumulation of APE1 through condensates is mediated by rRNA forming G-quadruplex structures

Abstract

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is the main endonuclease of the base excision repair (BER) pathway acting on abasic (AP)-sites in damaged DNA. APE1 is an abundant nuclear protein with a higher concentration than other BER pathway enzymes, and therefore, improper expression and localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 sub-cellular localization, expression levels, or hyper-acetylation are associated with cancer development suggesting the importance of a fine-tuning mechanism for APE1 nuclear-associated processes. Recent work highlighted multi-functional roles of APE1, including rRNA quality control. However, how rRNA influences the sub-cellular localization and activity of APE1 remains poorly understood, but previously underappreciated APE1-RNA interactions may influence the ability of this protein to form biomolecular condensates and tune APE1 partitioning into nucleoli. Since nucleolar accumulation of ectopic proteins could be the result of overexpression strategies, it is imperative to have cellular models to study APE1 trafficking under physiological conditions. Here we created the first cell line to express fluorescently tagged APE1 at its endogenous locus, enabling live-cell imaging. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription. When modeled in vitro, APE1 condensate formation depends on RNA G-quadruplex (rG4) structures in rRNA and is modulated by critical lysine residues of APE1. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and formation of RNA-dependent APE1 nucleolar condensates that may modulate a switch between the activity of this factor in rRNA processing and DNA damage repair. Significance StatementWe created and characterized the first endogenous, fluorescently tagged cell line to study APE1 subcellular trafficking under physiological and stress conditions. Using this cell line, we show that APE1 nucleolar enrichment occurs under physiological conditions and, performing in vitro droplet assays, we associate APE1 condensates with active transcription of RNA G-quadruplexes, abundantly present in healthy nucleoli. This work deepens our understanding of APE1s role in healthy cells in the absence of DNA damage and provide a novel mechanism for how this protein responds to stress. Our results suggest that phase separation is an important part of how DNA damage repair proteins switch between their normal physiological functions and their ability to correct DNA lesions.