title: RNA involvement during non-homologous end joining of resected DNA double strand breaks in G1
Creators: name: Wei, Na
Publisher: name: UNSPECIFIED
Year: 2022

description: DNA damage caused by physical or chemical mutagens threatens genomic integrity and the survival of living organisms. Particularly, DNA double-strand breaks (DSBs), the most hazardous lesions, arise when both DNA strands in the double helix are broken simultaneously, thus enhancing the risk of genome rearrangements. To revert the damaged genetic information and minimize the impact of damages in the genome, two major DSB repair pathways exist: non-homologous end joining (NHEJ) and homologous recombination (HR). In NHEJ, the two DSB ends are directly rejoined by DNA ligase IV complex and guided by short homologous DNA sequences in single-stranded overhangs at DSB ends. NHEJ accurately repairs DSBs only when these overhangs are perfectly matched, otherwise it can possibly lead to small insertions/deletions and translocations. Conversely, HR, only occurring during S/G2, utilizes the homologous sister-chromatid as a template to recombine the resected strands, thereby avoiding the loss of any information at DSBs. In fact, DSB repair kinetics consist of two components: the fast repair process which resolves most of the breaks within a few hours upon DSB induction, and the slow repair process that repairs the rest of breaks. Previous published work has characterized that the fast repair process in G1 and G2 both relies on resection-independent NHEJ, whereas the slow repair process represents resection-dependent NHEJ in G1 and HR in G2. However, why resection is required for the slow DSB repair in G1 remains unillustrated. Indeed, emerging evidence highlights that persistent DSBs in G1, when HR is unavailable, preferentially cluster at actively transcribed genes during the delayed repair. Besides, recent studies propose that transcription repression caused by DSB damage can be resumed at later times in actively transcribed regions. Therefore, it can be speculated that a correlation between transcription and resection may exist in the slow repair component in G1, thus being the main focus of this work. For this purpose, mouse embryonic stem cells (mESCs) were used, as they were found to be an interesting system to study resection. In fact, consistent with previous work, the co-immunostaining analysis of pRPA and γH2AX foci showed that resection also occurs in G1-mESCs at some X-ray irradiation (X-IR) and restriction enzyme-induced DSBs. Meanwhile, the reduction of pRPA foci formation when resection is inhibited by the PLK inhibitor (PLKi) and siCtIP, further confirms that a distinctive resection process arises in G1-mESCs, unlike the resection step in HR. Besides, inhibition of resection causes a repair defect, indicating that resection-dependent NHEJ is required for the repair of some DSBs in G1-mESCs. In addition, transcription inhibition reduces pRPA foci as well as DNA-RNA hybrid formation, and causes a repair defect in G1. However, the reduction of pRPA foci formation and the repair defect were both rescued when transcription was only inhibited before X-IR and resumed after X-IR. Taken together, these results suggest that transcription promotes DSB resection and is probably required for the slow DSB repair in G1. To investigate how resection affects transcription during DSB repair in G1, different EU pulse labeling methods were used to distinguish pre-existing RNA before DSB induction or damaged-induced RNA. The results showed that resection inhibition diminishes the accumulation of damaged-induced RNA but does not affect the pre-existing RNA, suggesting that resection may contribute to the resuming of transcription post DSB induction. Furthermore, DNA-RNA hybrid formation at DSBs was also observed in a resection-dependent manner. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) analysis suggests that DSBs which undergo resection in G1 are located in intragenic regions and that resection is required for the repair of those DSBs. Recent studies have also involved RAD52 in RNA-mediated DSB repair and showed that it was recruited to transcriptionally active damage sites during G0/G1 phase. Accordingly, we also observed that RAD52 is required for DNA-RNA hybrid formation at DSB sites. Strikingly, both immunofluorescence and ChIP-qPCR analysis showed that RAD52 deficiency does not affect the level of resection and does not cause a defect in the repair of intragenic DSBs in G1. This indicates that RAD52 is dispensable for initiating resection, but is likely to be a downstream factor that regulates DNA-RNA hybrid formation at DSBs. Collectively, these results suggest that resection-dependent NHEJ in G1 may preferentially arise in genes and be regulated by transcription. Moreover, RAD52 supports DNA-RNA hybrid formation at resected DSBs arising in genes which we hypothesize may enhance the repair fidelity.