Abstract
DNA integrity is constantly challenged by both endogenous and exogenous damaging agents, resulting in various forms of damage. Failure to repair DNA accurately leads to genomic instability, a hallmark of cancer. Distinct pathways exist to repair different types of DNA damage. Double-strand breaks (DSBs) represent a particularly severe form of damage, due to the physical separation of DNA strands. The repair of DSBs requires the activity of RNA Polymerase II (RNAPII) and the generation of Damage-responsive transcripts (DARTs). Here we show that the RNA m5C-methyltransferase NSUN2 localises to DSBs in a transcription-dependent manner, where it binds to and methylates DARTs. The depletion of NSUN2 results in an accumulation of nascent primary DARTs around DSBs. Furthermore, we detect an RNA-dependent interaction between NSUN2 and DICER, which is stimulated by DNA damage. NSUN2 activity promotes DICER cleavage of DARTs-associated R-loops, which is required for efficient DNA repair. We report a role of the RNA m5C -methyltransferase NSUN2 within the RNA-dependent DNA damage response, highlighting its function as a DICER chaperone for the clearance of non-canonical substrates such as DARTs, thereby contributing to genomic integrity.