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.异常R环（unscheduled R-loops）是复制应激与DNA损伤的核心来源。R环诱导的复制缺陷可被ATR激酶（ATR kinase）感知并抑制，但目前尚不明确R环本身是否主动参与ATR激酶的激活，若参与则具体机制如何。本研究发现，RNA编辑酶ADAR1的核定位形式能够促进ATR激酶激活，并清除全基因组范围内的R环，这一过程依赖其双链RNA结合结构域。从机制层面分析，ADAR1可与TOPBP1相互作用，并通过增强TOPBP1与9-1-1复合物中RAD9亚基的结合，促进TOPBP1在受扰动复制叉上的招募。当复制过程受到抑制时，DNA-RNA杂交链（DNA-RNA hybrid）会与TOPBP1竞争结合ADAR1，促使ADAR1从受损复制叉易位至R环区域。在此区域中，ADAR1可招募RNA解旋酶DHX9与DDX21以解旋R环，同时使TOPBP1能够更高效地激活ATR激酶。综上而言，本研究提出，受时空调控的以ADAR1为核心的蛋白质复合物组装，将R环清除与ATR激酶激活相耦联；而R环则通过转运ADAR1来微调ATR激酶活性、维护基因组稳态，以此与受阻复制叉发生串扰。