ADAR1 Links R-Loop Homeostasis to ATR Activation in Replication Stress Response

Unscheduled R-loops are a major source of replication stress and DNA damage. R-loop-induced replication defects are sensed and suppressed by ATR kinase, whereas it is not known whether R-loop itself is actively involved in ATR activation and, if so, how this is achieved. Here, we report that the nuclear form of RNA-editing enzyme ADAR1 promotes ATR activation and resolves genome-wide R-loops, a process that requires its double-stranded RNA-binding domains. Mechanistically, ADAR1 interacts with TOPBP1 and facilitates its loading on perturbed replication forks by enhancing the association of TOPBP1 with RAD9 of the 9-1-1 complex. When replication is inhibited, DNA-RNA hybrid competes with TOPBP1 for ADAR1 binding to promote the translocation of ADAR1 from damaged fork to R-loop region. There, ADAR1 recruits RNA helicases DHX9 and DDX21 to unwind R-loops, simultaneously allowing TOPBP1 to stimulate ATR more efficiently. Collectively, we propose that the tempo-spatially regulated assembly of ADAR1-nucleated protein complexes link R-loop clearance and ATR activation, while R-loops crosstalk with blocked replication forks by transposing ADAR1 to finetune ATR activity and safeguard the genome. The genome extracted from Hela cells expressing control siRNA or ADAR1 siRNA were treated with or without RNase H, then incubated with S9.6 antibody for immunoprecipitation and sequencing.