CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

Abstract

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we develop single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and helps re-activate RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

RNA Polymerase II-mediated transcription is required for repair of ribosomal DNA breaks in nucleolar caps, guarding against genomic instability

“…3C and D). These results suggest that early transcription steps do occur in the vicinity of the rDNA DSBs at the periphery of the nucleolus, consistent with a functional link between active RNAPII with rDNA repair by HR (9). Finally, we analysed the profile for other phosphorylations in the CTD of the RNAPII, specifically in Tyrosine 1 (RNAPII-Y1P), which some studies have related it to DSB repair (26).…”

“…RNA transcription associated with DNA repair by HR is an essential aspect of accurate DSB repair in nucleoplasm (9). Ribosomal DNA breaks move out to the nucleoli, specifically to the nucleolar caps where HR can repair them with high fidelity, thereby avoiding cis-recombination between rDNA copies to preserve the stability of these vulnerable genomic regions.…”

“…1D) (9). The reason why THZ1 treatment also partly reduces DNA resection in cells transfected by an empty vector instead of gRNA reflects the fact that RNAPII also plays a role in repair of endogenous DNA damage (9). Monitoring formation of NBS1-GFP-containing nucleolar caps after transfection of rDNA-specific gRNAs demonstrated that more than 25% of cells accumulated these structures in the nucleolar periphery by 6 hours after gRNA transfection (Fig.…”

“…2B, C and D). To minimize the likelihood of any off-target effect of THZ1, we used another known RNAPII inhibitor, triptolide, that affects RNA transcription initiation by RNAPII degradation via proteasome (9,23). We observed similar nucleolar cap formation deficiency after triptolide treatment, demonstrating that the inhibition of transcription initiation by RNAPII indeed leads to nucleolar cap formation (Fig.…”

“…Due to such 'nucleolar stress', rDNA instability occurs commonly during tumorigenesis, likely fueling tumor progression due to rDNA rearrangements events including insertions, translocations and amplifications (8). Among the different rDNA damage arising from transcription-replication conflicts, the rDNA double-strand breaks (rDNA DSBs) are arguably the most cytotoxic and difficult to repair lesions (9)(10)(11). The two main pathways for DSB repair are homologous recombination (HR) and non-homologous end joining (NHEJ), both of which contribute to rDNA stability upon oncogenic or genotoxic stresses, such as activated cMYC oncogene and ionizing radiation (IR), respectively (11,12).…”