RNA damage induced DNA breaks: A new mechanism of genome instability

The major goal of this proposal is to define the mechanisms of a new pathway for genome stability, which we call RDIBs (RNA damage induced DNA breaks). It has been known for decades that many DNA damaging agents also damage RNA, due to their similar chemistries. However, outside of RNA quality control mechanisms related to translational control in the cytoplasm, the direct implications of RNA damage for genome stability in the nucleus are largely unknown. Our new data indicates that damage to nascent RNA through base methylation, if not promptly removed, results in DNA double-stranded breaks. Through a targeted screen for factors that recognize methylated RNA, we identify the RNA binding protein YTHDC1 as being critical for alkylation damage responses in conjunction with the THO complex (THOC). In the absence of YTHDC1 or THOC, alkylation base damage results in greater damage sensitivity and DNA breaks. This damage is fully attributable to RNA damage, since an RNA-specific demethylase can rescue these phenotypes. These breaks depend on R-loop formation, which are then processed by a structure-specific nuclease. We also show that in the absence of YTHDC1 or THOC, aberrant RNA methylation in the nucleus is sufficient to induce DNA breaks near the RNA methylation site. Our discovery of this pathway provides definitive evidence for how RNA damage can impact genomic integrity. Yet, several key questions about the RDIBs pathway remain, which we seek to answer in this proposal. We will define the causes and consequences of RNA damage induced breaks, by identifying the types of RNA modifications that activate RDIBs, the structure of the DNA break, the nucleases responsible for their formation, and the mechanism for the repair of the ensuing break (Aim 1). In parallel, we will use cell biological and biochemical reconstitution approaches to demonstrate how YTHDC1-THOC, along with an associated helicase and RNA endonuclease, help to resolve damage to nascent RNA in the context of R-loops (Aim 2). Together, these studies will shed light on an undiscovered pathway by which RNA damage can cause loss of genome integrity, as well as the mechanisms that suppress this form of damage.