SignificanceTranscription by RNA Polymerase II (Pol II) is a highly dynamic process that is tightly regulated at each step of the transcription cycle. We generated GFP-RPB1 knockin cells and developed photobleaching of endogenous Pol II combined with computational modeling to study the in vivo dynamics of Pol II in real time. This approach allowed us to dissect promoter-paused Pol II from initiating and elongating Pol II and showed that initiation and promoter proximal pausing are surprisingly dynamic events, due to premature termination of Pol II. Our study provides new insights into Pol II dynamics and suggests that the iterative release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda−/−) mouse-model resembling TTD-A patients. Unexpectedly, Ttda−/− mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda−/− cells was not affected. Surprisingly, Ttda−/− cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda−/− cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda−/− cells as a unique class of TFIIH mutants.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

Highlights d RNAPII S5 phosphatase PNUTS-PP1 promotes RNAPII turnover on chromatin d Depletion of PNUTS leads to transcription-replication conflicts d WDR82 shows similar effects on RNAPII turnover and replication stress as PNUTS d CDC73 prevents RNAPII degradation and promotes replication stress after PNUTS depletion

The faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The stalling of RNAP2 on a transcription-blocking lesion triggers a series of highly regulated events, including RNAP2 processing to make the lesion accessible for DNA repair, R-loop-mediated DNA damage signaling, and the initiation of transcription-coupled DNA repair. The correct execution and coordination of these processes is vital for resuming transcription following the successful repair of transcription-blocking lesions. Here, we outline recent insights into the molecular consequences of RNAP2 stalling on transcription-blocking DNA lesions and how these lesions are resolved to restore mRNA synthesis.

The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress.

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1—proteins involved in the signaling axis of mammalian DSB repair—in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.