ATR phosphorylates DHX9 at serine 321 to suppress R-loop accumulation upon genotoxic stress.

Aberrant DNA/RNA hybrids (R-loops) formed during transcription and replication disturbances pose threats to genome stability. DHX9 is an RNA helicase involved in R-loop resolution, but how DHX9 is regulated in response to genotoxic stress remains unclear. Here we report that DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation primarily induced by ATR after DNA damage. Phosphorylation of DHX9 at S321 promotes its interaction with γH2AX, BRCA1 and RPA, and is required for its association with R-loops under genotoxic stress. Inhibition of ATR or expression of the non-phosphorylatable DHX9S321A prevents DHX9 from interacting with RPA and R-loops, leading to the accumulation of stress-induced R-loops. Furthermore, depletion of RPA reduces the association between DHX9 and γH2AX, and in vitro binding analysis confirms a direct interaction between DHX9 and RPA. Notably, cells with the non-phosphorylatable DHX9S321A variant exhibit hypersensitivity to genotoxic stress, while those expressing the phosphomimetic DHX9S321D variant prevent R-loop accumulation and display resistance to DNA damage agents. In summary, we uncover a new mechanism by which ATR directly regulates DHX9 through phosphorylation to eliminate stress-induced R-loops.