scholarly journals ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

2021 ◽  
Author(s):  
Yana van der Weegen ◽  
Klaas de Lint ◽  
Diana van den Heuvel ◽  
Yuka Nakazawa ◽  
Ishwarya V. Narayanan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Genetic Interaction ◽  
Dna Lesions ◽  
Specificity Factor ◽  
Interaction Screening ◽  
Repair Factor ◽  
Transcription And Replication

SummaryCells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. The binding of the TCR-specific repair factor CSB triggers DNA damage-induced ubiquitylation of RNA polymerase II (RNAPII) at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. However, how the CRL4CSA ligase is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation. Furthermore, drug-genetic interaction screening reveals an unanticipated compensatory TCR pathway in which ELOF1 together with known factors DOT1L and HIRA protect CSB-deficient cells from collisions between transcription and replication machineries. Our study provides a genetic framework of the transcription stress response and reveals key insights into the molecular mechanism of TCR.

scholarly journals DNA damage stabilizes interaction of CSB with the transcription elongation machinery

2004 ◽  
Vol 166 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Vincent van den Boom ◽  
Elisabetta Citterio ◽  
Deborah Hoogstraten ◽  
Angelika Zotter ◽  
Jean-Marc Egly ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Dna Damage ◽  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Molecular Weight ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
High Molecular Weight Complex

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

scholarly journals Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair

2019 ◽  
Author(s):  
Katja Apelt ◽  
Iris Zoutendijk ◽  
Dennis Y. Gout ◽  
Diana van den Heuvel ◽  
Martijn S. Luijsterburg
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Light ◽  
Dna Lesions ◽  
Illudin S ◽  
Highly Sensitive ◽  
Nucleosome Binding ◽  
Active Genes

SummaryTranscription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR – particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions were GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.

scholarly journals A SUMO-dependent pathway controls elongating RNA Polymerase II upon UV-induced damage

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Irina Heckmann ◽  
Maximilian J. Kern ◽  
Boris Pfander ◽  
Stefan Jentsch
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Rna Polymerase Iii ◽  
Messenger Rnas ◽  
Eukaryotic Transcription ◽  
Small Nuclear Rnas ◽  
Defective Rna ◽  
Dependent Pathway

AbstractRNA polymerase II (RNAPII) is the workhorse of eukaryotic transcription and produces messenger RNAs and small nuclear RNAs. Stalling of RNAPII caused by transcription obstacles such as DNA damage threatens functional gene expression and is linked to transcription-coupled DNA repair. To restore transcription, persistently stalled RNAPII can be disassembled and removed from chromatin. This process involves several ubiquitin ligases that have been implicated in RNAPII ubiquitylation and proteasomal degradation. Transcription by RNAPII is heavily controlled by phosphorylation of the C-terminal domain of its largest subunit Rpb1. Here, we show that the elongating form of Rpb1, marked by S2 phosphorylation, is specifically controlled upon UV-induced DNA damage. Regulation of S2-phosphorylated Rpb1 is mediated by SUMOylation, the SUMO-targeted ubiquitin ligase Slx5-Slx8, the Cdc48 segregase as well as the proteasome. Our data suggest an RNAPII control pathway with striking parallels to known disassembly mechanisms acting on defective RNA polymerase III.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

2021 ◽  
Author(s):  
Priyanka Barman ◽  
Rwik Sen ◽  
Amala Kaja ◽  
Jannatul Ferdoush ◽  
Shalini Guha ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Initiation Complex ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Genome Wide

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

scholarly journals RNA polymerase II transcription is concentrated outside replication domains throughout S-phase

1994 ◽  
Vol 107 (6) ◽  
pp. 1449-1456 ◽  
Author(s):  
D.G. Wansink ◽  
E.E. Manders ◽  
I. van der Kraan ◽  
J.A. Aten ◽  
R. van Driel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Relationship ◽  
S Phase ◽  
Double Labelling ◽  
Confocal Immunofluorescence Microscopy ◽  
Nuclear Domain ◽  
Confocal Immunofluorescence ◽  
Nuclear Domains ◽  
Transcription And Replication

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

scholarly journals Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

2014 ◽  
Vol 35 (2) ◽  
pp. 468-478 ◽  
Author(s):  
Tristan T. Eifler ◽  
Wei Shao ◽  
Koen Bartholomeeusen ◽  
Koh Fujinaga ◽  
Stefanie Jäger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Specificity Factor

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3′ end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

1992 ◽  
Vol 12 (9) ◽  
pp. 4142-4152
Author(s):  
J Archambault ◽  
F Lacroute ◽  
A Ruet ◽  
J D Friesen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Integrity ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Chemical Mutagenesis ◽  
Yeast Genome ◽  
Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

scholarly journals GSK-3 is an RNA polymerase II phospho-CTD kinase

2020 ◽  
Vol 48 (11) ◽  
pp. 6068-6080 ◽  
Author(s):  
Nicolás Nieto Moreno ◽  
Florencia Villafañez ◽  
Luciana E Giono ◽  
Carmen Cuenca ◽  
Gastón Soria ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Glycogen Synthase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transcriptional Elongation ◽  
Induced Apoptosis ◽  
Carboxy Terminal

Abstract We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

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