ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop

Abstract

R loops are three-stranded transcription intermediates consisting of RNA:DNA hybrids and displaced single-stranded DNA. Although R loop formation is important for a number of cellular processes, they can cause genomic instability if dysregulated. The R loop-induced genomic instability is largely dependent on DNA replication, suggesting that it may arise from the collisions between R loops and DNA replication forks. How cells respond to the collisions between R loops and replication fork is poorly understood. My study reveals that dysregulated R loop formation during DNA replication activates the ATR checkpoint kinase, the master regulator of the replication stress response. The activation of ATR by aberrant R loops requires reversal of DNA replication forks and DNA cleavage by the MUS81 endonuclease. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation is uniquely dependent upon MUS81. Once activated, ATR protects the genome from R loops by suppressing transcription-replication collisions, enforcing a G2/M arrest, and promoting fork recovery. Additionally, ATR prevents excessive cleavage of reversed forks by MUS81, suggesting that MUS81 triggers a feedback loop in which ATR regulates MUS81 activity at replication forks. Overall, these results reveal an ATR-regulated signaling circuitry induced by collisions of R loops and replication forks, and establish ATR as a key sensor and suppressor of R loop-induced genomic instability.