Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/proteasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.

Nucleotide excision repair (NER) guarantees genome integrity against UV light-induced DNA damage. After UV irradiation, cells have to cope with a general transcriptional block. To ensure UV lesions repair specifically on transcribed genes, NER is coupled with transcription in an extremely organized pathway known as transcription-coupled repair. In highly metabolic cells, more than 60% of total cellular transcription results from RNA polymerase I activity. Repair of the mammalian transcribed ribosomal DNA has been scarcely studied. UV lesions severely block RNA polymerase I activity and the full transcription-coupled repair machinery corrects damage on actively transcribed ribosomal DNAs. After UV irradiation, RNA polymerase I is more bound to the ribosomal DNA and both are displaced to the nucleolar periphery. Importantly, the reentry of RNA polymerase I and the ribosomal DNA is dependent on the presence of UV lesions on DNA and independent of transcription restart.

Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery but little is known regarding the role of each Mediator subunits. Mutations in MED12 are linked with a broad spectrum of genetic disorders with X-linked intellectual disability that are difficult to range as Lujan, Opitz-Kaveggia or Ohdo syndromes. Here, we investigated several MED12 patients mutations (p.R206Q, p.N898D, p.R961W, p.N1007S, p.R1148H, p.S1165P and p.R1295H) and show that each MED12 mutations cause specific expression patterns of JUN, FOS and EGR1 immediate early genes (IEGs), reflected by the presence or absence of MED12 containing complex at their respective promoters. Moreover, the effect of MED12 mutations has cell-type specificity on IEG expression. As a consequence, the expression of late responsive genes such as the matrix metalloproteinase-3 and the RE1 silencing transcription factor implicated respectively in neural plasticity and the specific expression of neuronal genes is disturbed as documented for MED12/p.R1295H mutation. In such case, JUN and FOS failed to be properly recruited at their AP1-binding site. Our results suggest that the differences between MED12-related phenotypes are essentially the result of distinct IEGs expression patterns, the later ones depending on the accurate formation of the transcription initiation complex. This might challenge clinicians to rethink the traditional syndromes boundaries and to include genetic criterion in patients' diagnostic.

During DNA Repair, ribosomal DNA and RNA polymerase I (rDNA/RNAP1) are reorganized within the nucleolus. Until now, the proteins and the molecular mechanism governing this reorganisation remained unknown.Here we show that Nuclear Myosin I (NMI) and Nuclear Beta Actin (ACTβ) are essential for the proper reorganisation of the nucleolus, after completion of the DNA Repair reaction.In NMI and ACTβ depleted cells, the rDNA/RNAP1 complex can be displaced at the periphery of the nucleolus after DNA damage but cannot re-enter within the nucleolus after completion of the DNA Repair. Both proteins act concertedly in this process. NMI binds the damaged rDNA at the periphery of the nucleolus, while ACTβ brings the rDNA back within the nucleolus after DNA repair completion.Our results reveal a previously unidentified function for NMI and ACTβ and disclose how these two proteins work in coordination to re-establish the proper rDNA position after DNA repair.

DNA lesions block cellular processes such as transcription, inducing apoptosis, tissue failures, and premature aging. To counteract the deleterious effects of DNA damage, cells are equipped with various DNA repair pathways. Transcription-coupled repair specifically removes helix-distorting DNA adducts in a coordinated multistep process. This process has been extensively studied; however, once the repair reaction is accomplished, little is known about how transcription restarts. In this study, we show that, after UV irradiation, the cyclin-dependent kinase 9 (CDK9)/cyclin T1 kinase unit is specifically released from the HEXIM1 complex and that this released fraction is degraded in the absence of the Cockayne syndrome group B protein (CSB). We determine that UV irradiation induces a specific Ser2 phosphorylation of the RNA polymerase II and that this phosphorylation is CSB dependent. Surprisingly, CDK9 is not responsible for this phosphorylation but instead might play a nonenzymatic role in transcription restart after DNA repair.

NUCLEOTIDE EXCISION REPAIR; RNAP2: RNA POLYMERASE II 24 25 26 2 AUTHOR SUMMARY 27 DNA lesions block cellular processes such as transcription, inducing apoptosis, tissue failures and 28 premature ageing. To counteract the deleterious effects of DNA damage, cells are equipped with 29 various DNA repair pathways. Transcription Coupled Repair specifically removes helix--distorting DNA 30adducts in a coordinated multi--step process. This process has been extensively studied, however 31 once the repair reaction is accomplished, little is known about how transcription restarts. In this 32 study, we show that, after UV irradiation, the CDK9/CyclinT1 kinase unit is specifically released from 33 the HEXIM1 complex and that this released fraction is degraded in the absence of CSB. We determine 34 that UV--irradiation induces a specific Ser2 phosphorylation of the RNA polymerase II and that this 35 phosphorylation is CSB dependent. Surprisingly CDK9 is not responsible for this phosphorylation but 36 instead plays a non--enzymatic role in transcription restart after DNA repair. 37 38 3 Introduction 39 Cells are the units of organic life and store in their nuclei, under the form of the DNA 40 molecule, what constitutes the instruction manual for proper cellular functioning. Despite the 41 protection offered by the cellular environment, the integrity of DNA is continuously challenged by a 42 variety of endogenous and exogenous agents (e.g. ultraviolet light, cigarette smoke, environmental 43 pollution, oxidative damage, etc …) that cause DNA lesions, interfering with proper cellular functions, 44 in fine causing the aging or premature aging of the tissue and later on of the whole organism.45 To prevent the deleterious consequences of persisting DNA lesions, all organisms are 46 equipped with a network of efficient DNA damage responses and DNA repair systems. One of these 47 systems is the Nucleotide Excision Repair (NER). NER removes helix--distorting DNA adducts such as 48 UV--induced lesions (Cyclo--Pyrimidine Dimers and 6--4 Photoproducts, CPD and 6--4PP) in a 49 coordinated multi--step process (1). 50The NER system has been linked to rare human diseases classically grouped into three 51 distinct NER--related syndromes. These include the highly cancer prone disorder xeroderma 52 pigmentosum (XP) and the two progeroid diseases: Cockayne syndrome (CS) and trichothiodystrophy 53 (TTD) (2). Importantly, CS and TTD patients are not cancer--prone but present severe neurological and 54 developmental features. 55NER exists in two distinct sub--pathways depending where DNA lesions are located within the 56 genome. Global Genome Repair (GG--NER or GGR) will repair DNA lesion located on non--transcribed 57 DNA. While, the second sub--pathway is directly coupled to transcription elongation and repairs DNA 58 lesions located on the transcribed strand of active genes and it is designated as Transcription-- 59Coupled Repair (TC--NER or TCR). 60RNAP2 frequently deals with obstacles that need to be removed through the TCR process for 61 resu...

Background The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway. Methods We used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues. Results Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell’s transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. Conclusion This study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation.

Xeroderma Pigmentosum group A-binding protein 2 (XAB2) is a multi-functional protein playing a critical role in distinct cellular processes including transcription, splicing, DNA repair and mRNA export. In this study, we demonstrate that XAB2 is involved specifically and exclusively in Transcription-Coupled Nucleotide Excision Repair (TC-NER) reactions and solely for RNA Polymerase 2 transcribed genes. Surprisingly, contrary to all the other NER proteins studied so far, XAB2 does not accumulate on the local UV-C damage; on the contrary, it becomes more mobile after damage induction. XAB2 mobility is restored when DNA repair reactions are completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have identified that XAB2 is detached after DNA damage induction from DNA:RNA hybrids, commonly known as R-loops, and from the CSA and XPG proteins. This release contributes to the DNA damage recognition step during TC-NER, as in the absence of XAB2, RNAP2 is blocked longer on UV lesions. Moreover, we also demonstrate that XAB2 has a role in retaining RNAP2 on its substrate without any DNA damage.