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Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell–imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process. DNA damage sensors DDB2 and XPC are fundamental factors to initiate global genome nucleotide excision repair and protect DNA from mutagenesis. Here the authors reveal that ubiquitin and TFIIH-stimulated DDB2 dissociation promotes DNA damage handover to XPC in nucleotide excision repair.

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions. Nucleotide excision repair proteins are involved in the repair of UV-induced DNA damage. Here, the authors show that NER proteins, DDB2, XPC, and XPA play a vital role in the 8-oxoguanine repair by coordinating with base excision repair protein OGG1.

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. DNA damage inhibits elongating RNA polymerase II, but also initiates genome-wide transcriptional responses. Here the authors reveal that particularly promoter-bound Pol II is degraded upon DNA damage in a GSK3 signaling-mediated response.

XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4 DDB2 ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER. The SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. Here the authors show that RNF111 is responsible for sequential XPC ubiquitylation, and RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation.

Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans , this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA. Hereditary nucleotide excision repair deficiencies cause different cancerous and progeroid disorders of which the exact etiology is not understood. This study finds that prolonged binding of DNA repair factor TFIIH to DNA damage contributes to a more severe phenotype caused by DNA repair deficiency.

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4 CSA ). Although ubiquitination of several TC-NER proteins by CRL4 CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4 CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process. Transcription-Coupled Nucleotide Excision Repair (TC-NER) removes transcription-blocking DNA lesions. This study reveals that DDA1 is a crucial player in TC-NER, protecting transcription programs against genotoxic insults.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD‐associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady‐state mutant protein levels. However, to date, no such link to instability of gene‐expression factors for TTD‐associated mutations in MPLKIP / TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry‐based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP ‐deficient primary fibroblasts have reduced steady‐state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue. Synopsis MPLKIP‐deficiency is one of the causative genes for trichothiodystrophy (TTD), a condition characterized by clinical features associated with ectodermal abnormalities. We used mass spectrometry‐based interaction proteomics to disclose the still unknown biological function of MPLKIP and applied a Human 3D skin model to investigate its role in skin development. MPLKIP is a protein associated with pre‐mRNA splicing and plays a crucial role in maintaining cellular protein levels of its complex partner DBR1. Human skin models lacking MPLKIP display “leaky” epithelial barrier formation with a reduced epidermal thickness and impaired immune response. MPLKIP‐deficiency results in abnormal pre‐mRNA splicing, accumulation of lariat introns, and altered protein expression. Graphical Abstract MPLKIP‐deficiency is one of the causative genes for trichothiodystrophy (TTD), a condition characterized by clinical features associated with ectodermal abnormalities. We used mass spectrometry‐based interaction proteomics to disclose the still unknown biological function of MPLKIP and applied a Human 3D skin model to investigate its role in skin development.

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.

DNA damage and repair are central to the onset of cancer, aging, and aging-related diseases. Rare genetic defects in the nucleotide excision repair pathway, such as those causing the cancer-prone disorder xeroderma pigmentosum (XP) or the progeroid condition Cockayne syndrome, highlight the dramatic consequences of unrepaired DNA lesions. In this issue of the JCI, two related papers from Ogi and coworkers - Fassihi et al. and Nakazawa et al. - describe a new XP clinical entity, XP-J, linked to a pathogenic variant in the p52 subunit of the transcription-repair complex TFIIH. The studies' characterization of XP-J and the p52ΔC variant opened unexpected possibilities to ameliorate the molecular defect in another subunit of TFIIH that causes a different, more severe repair syndrome: trichothiodystrophy. This commentary provides a broader historical, medical, and molecular context for the intricate genotype-phenotype relationship between compromised repair and its clinical consequences and discusses next steps for the advances reported.