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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Aberrant DNA/RNA hybrids (R-loops) arise from transcription and replication conflicts, compromising genome stability.
• The RNA helicase DHX9 resolves R-loops, but its regulation under genotoxic stress is not fully understood.

Purpose of the Study:

• To elucidate the regulatory mechanism of DHX9 in response to genotoxic stress.
• To investigate the role of DHX9 phosphorylation in R-loop resolution and genome stability.

Main Methods:

• Western blotting to detect DHX9 phosphorylation.
• Co-immunoprecipitation to assess protein interactions (DHX9, γH2AX, BRCA1, RPA).
• Analysis of R-loop accumulation using microscopy and molecular assays.
• Site-directed mutagenesis to create non-phosphorylatable (S321A) and phosphomimetic (S321D) DHX9 variants.
• Cellular sensitivity assays to genotoxic agents.

Main Results:

• DHX9 is phosphorylated at S321 by ATR kinase upon DNA damage.
• S321 phosphorylation enhances DHX9 binding to γH2AX, BRCA1, and RPA, facilitating R-loop association.
• ATR inhibition or S321A mutation impairs DHX9-RPA interaction and R-loop resolution, increasing R-loop accumulation.
• RPA depletion reduces DHX9-γH2AX association.
• DHX9S321A cells show hypersensitivity to genotoxic stress, while DHX9S321D cells exhibit resistance.

Conclusions:

• ATR-mediated phosphorylation of DHX9 at S321 is a critical regulatory mechanism for resolving stress-induced R-loops.
• This phosphorylation event is essential for DHX9 recruitment of RPA and subsequent R-loop resolution, maintaining genome stability.