Ubiquitylation-mediated RNAPII genomic eviction in response to DNA DSBs
Essential biological functions are constantly challenged by genotoxic stresses. During transcription, DNA Damage can occur at any stage predisposing genomic stability through the direct damage to DNA and formation of aberrant/truncated transcripts. A central surveillance mechanism is operated by R...
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| Dokumentumtípus: | Disszertáció |
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2024-11-22
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| Online Access: | http://doktori.ek.szte.hu/12363 |
| Tartalmi kivonat: | Essential biological functions are constantly challenged by genotoxic stresses. During transcription, DNA Damage can occur at any stage predisposing genomic stability through the direct damage to DNA and formation of aberrant/truncated transcripts. A central surveillance mechanism is operated by RNAPII when it encounters a DNA lesion. If the lesion persists or is difficult to repair as in the case of DSBs (DSBs) then the RNAPII must be removed from the damaged site to allow for DNA repair. The removal and processing of RNAPII is surprisingly a ubiquitylation-dependent mechanism mediated by ubiquitin ligases. Although in recent years different E3 ubiquitin ligases have emerged as the preferred choices of big pharmaceutical and biotechnological startups in the context of cancer and disease biology, it appears that the E3 ubiquitin ligases described in this project (WWP2, NEDD4 and CRL3 ubiquitin complex), are not very well recognized yet in the realm of molecular biology and drug discovery. In this project, we were trying to elucidate the mechanisms of ubiquitylation and identify the biological complexes involved in DSB-induced transcription stress response. The elegance of our designed investigation using novel established practices in our laboratory (LC-MS/MS, TUBE pull downs, UbiCRest, ChIPs etc.) let us uncover that the ubiquitylation of the transcribing RNAPII upon DSB encounter is proteasomal dependent mediated mainly by, NEDD4 and CUL3 complexes in a timedependent fashion while WWP2 depends on a mechanism that does not involve the proteasome. We are able to distinguish WWP2 as the initial responder to DSBs, while NEDD4 proves indispensable in the subsequent stages, during break persistence ensuring accurate repair and genome integrity. In our experimental setup CUL3 ubiquitin complexes exhibit an auxiliary yet important role in the clearance of RNAPII altering the transcription cycle and dynamics upon its depletion. Although it could only interact with the elongating RNAPII during persistent break induction its ubiquitylation had minimal impact upon CUL3 knock down. On the other hand, we observed greater impact on the ubiquitylation in physiological conditions where we could not observe any direct/indirect interaction of the two proteins. We believe that these findings suggest that CRL3 complexes are members of physiological recycling of the elongating RNAPII affecting the process indirectly whereas during DSBs it hasa yet to be uncovered role that facilitates transcription repression though not by direct ubiquitylation of RNAPII. Strikingly, we further observe the presence of K63 ubiquitin chains written on the elongating RNAPII substrate which were previously characterised to be mainly involved during regulation of DNA repair. We further demonstrate that these K63 chains are predominantly written by NEDD4 ligase. On the other hand, when it comes to the RNAPII holoenzyme K48 chains see to dominate and extend the K63 ones. Therefore, we posit that the ubiquitylation of elongating RNAPII constitutes a critical mechanism in the DSB repair pathway. This hypothesis was subsequently validated by demonstrating reduced NHEJ repair following knock-down of the ligases. We further confirm that the ligases are indeed recruited in the DSB site in a DNA-PK dependant manner and that the elongating RNAPII keeps transcribing a damaged unit when the ligases are knocked down, further substantiating our results (Figure 19). All in all, our project uncovered in depth the consequential links between DSB-induced transcriptional silencing, DSB repair, and ubiquitylation and according to this, we hope to unravel key mechanisms that contribute to the pathogenesis of cancer and provide strong basis of targeted cancer therapies. |
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