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CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway
Nature Communications volume 13, Article number: 5303(2022) Cite this article
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.
The concept of an RNA world postulates that RNA was essential for molecular processes and biochemical reactions implicated in the origin of life on Earth1. To compensate for RNA instability, DNA appeared later during the evolution to better preserve genetic information, followed by fidelity mechanisms to maintain genome stability1. Recently, RNA has emerged as a major factor in essential mechanisms regulating gene expression2,3 and contributing actively to DNA repair processes4,5,6,7,8,9. Arguably the most cytotoxic genomic lesions are DNA double-strand breaks (DSBs), lesions repaired mainly by either of the two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR)10,11,12,13,14. While numerous protein components of these two pathways have been discovered over time, only recently RNA has been implicated in DSB repair as well. For example, recent evidence showed that DSBs in transcriptionally active genomic regions are more prone to be repaired by HR7,8,15.
Interestingly, only 2-8% of the human genome gets transcribed16, yet it is unclear, unlikely perhaps, that HR is restricted to these genomic regions only. Hence, chromatin structure at transcriptionally active sites and the influence of diverse, relevant mechanisms, including DNA repair pathways, are currently subject to intense investigation to elucidate to what extent and how RNA impacts DSB repair. So far, such efforts generated controversial results. On the one hand, global RNA transcription is inhibited after DNA damage to avoid conflicts between repair and other DNA metabolic processes such as replication17. RNA involvement in DNA repair processes has also been demonstrated to contribute to genomic instability by forming RNA:DNA hybrid structures4,18.
On the other hand, the formation of RNA:DNA hybrids regulates DNA repair in diverse organisms7,8,19, exemplified by the DNA damage response RNAs (DDRNAs), necessary for DDR activation20,21,22. Furthermore, recruitment of general transcription factors to DNA damage sites was reported, and an active role for RNA polymerase II (RNAPII) in DNA repair was proposed23. Recently, RNA polymerase III was reported to be actively recruited to DSBs by the MRN complex and mediate RNA synthesis, promoting HR repair24. However, any role of RNAPII, the major mammalian RNA polymerase, in this context remains unknown. Any potential mechanistic contribution to DNA repair pathway choice could also inspire cancer treatment strategies to be combined with standard-of-care DNA damaging radio-chemotherapy.
As the repair mode is critical for genomic integrity and thereby inheritance, evolution, organismal development, and tissue homeostasis, the emerging evidence for RNA involvement raises the crucial questions of whether and how could RNA guide the choice between HR and NHEJ in DSB repair, an issue that we address in our present study.
Here, we elucidate the role played by de novo RNA synthesis by RNAPII in DSB repair via HR in human cells. We found that RNA presence during different cell cycle phases impacts the decision between the HR and NHEJ repair pathways, combined with the homologous sequences of sister chromatids. Using our single-molecule analysis approaches, we observed nascent RNA overlapping with ssDNA resection tracts generated during DNA end resection, indicating that RNAs, mainly synthesized by RNAPII, are essential to initiate DNA end resection and thereby shift the choice of DSB repair towards using the more faithful HR over NHEJ. Indeed, RNA:DNA hybrid formation is essential for resection processing and as a repair regulatory step in the HR pathway. Moreover, RNAPII inhibition, using a specific CDK7 inhibitor THZ1, impairs HR factor recruitment to DSB. We further demonstrate a previously unsuspected function of CtIP as a transcription re-activator of RNAPII paused transiently by the RNA:DNA hybrids during the early stage response to DSBs, thereby promoting DNA resection and skewing DSB repair balance towards HR.