scholarly journals CSB-dependent CDK9 degradation and RNA Polymerase II phosphorylation during Transcription Coupled Repair

2018 ◽  
Author(s):  
Lise-Marie Donnio ◽  
Anna Lagarou ◽  
Gabrielle Sueur ◽  
Pierre-Olivier Mari ◽  
Giuseppina Giglia-Mari
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Irradiation ◽  
Dna Adducts ◽  
Dna Lesions ◽  
Step Process ◽  
Cellular Processes ◽  
Repair Pathways ◽  
Transcription Coupled Repair

AUTHOR SUMMARYDNA lesions block cellular processes such as transcription, inducing apoptosis, tissue failures and premature ageing. 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 multi-step 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 CDK9/CyclinT1 kinase unit is specifically released from the HEXIM1 complex and that this released fraction is degraded in the absence of 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 plays a non-enzymatic role in transcription restart after DNA repair.

scholarly journals CSB-Dependent Cyclin-Dependent Kinase 9 Degradation and RNA Polymerase II Phosphorylation during Transcription-Coupled Repair

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Lise-Marie Donnio ◽  
Anna Lagarou ◽  
Gabrielle Sueur ◽  
Pierre-Olivier Mari ◽  
Giuseppina Giglia-Mari
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Irradiation ◽  
Dna Lesions ◽  
Premature Aging ◽  
Cyclin Dependent Kinase ◽  
Cyclin T1 ◽  
Group B ◽  
Transcription Coupled Repair

ABSTRACT 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.

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 Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chunli Yan ◽  
Thomas Dodd ◽  
Jina Yu ◽  
Bernice Leung ◽  
Jun Xu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Bound States ◽  
Dna Lesions ◽  
Functional Interpretation ◽  
Structural Mechanism ◽  
Disease Mutations ◽  
Dynamic Communities ◽  
Allosteric Pathway ◽  
Transcription Coupled Repair

AbstractTranscription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

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 Elevated transcriptional pausing of RNA polymerase II underlies acquired resistance to radiotherapy

2021 ◽  
Author(s):  
Honglu Liu ◽  
Chunhong Yu ◽  
Na Zhang ◽  
Yang Meng ◽  
Canhua Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Acquired Resistance ◽  
Dna Lesions ◽  
Steady Increase ◽  
Cellular Processes ◽  
Genome Wide ◽  
Transcriptional Pausing ◽  
Xenograft Models ◽  
Resistant Cells

As the mainstay modality for many malignancies, particularly inoperable solid tumors such as nasopharyngeal carcinoma (NPC), ionizing radiation (IR) induces a variety of lesions in genomic DNA, evoking a multipronged DNA damage response to interrupt many cellular processes including transcription. The turbulence in transcription, depending on the nature of DNA lesions, encompasses local blockage of RNA polymerase II (RNAPII) near the damage sites, as well as a less understood genome-wide alteration. How the transcriptional change influences the effectiveness of radiotherapy remains unclear. Using a panel of NPC and lung cancer cell lines, we observe increased phosphorylation at serine 5 (pS5) of the RNAPII after IR, indicating an accumulation of paused RNAPII. Remarkably, a similar increase of pS5 is seen in IR-resistant cells. ChIP-seq analysis of RNAPII distribution confirms this increased pausing both in IR-treated and IR-resistant NPC cells, notably on genes involved in radiation response and cell cycle. Accordingly, many of these genes show downregulated transcripts abundance in IR-resistant cells, and individual knockdown some of them such as TP53 and NEK7 endows NPC cells with varying degrees of IR-resistance. Decreasing pS5 of RNAPII and hence tuning down transcriptional pausing by inhibiting CDK7 reverses IR-resistance both in cell culture and xenograft models. Our results therefore uncover an unexpected link between elevated transcriptional pausing and IR-resistance. Given the recurrent NPC tissues display a steady increase in pS5 compared to the paired primary tissues, we suggest that CDK7 inhibitors can be used in combination with radiotherapy to increase sensitivity and thwart resistance.

scholarly journals Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e72090 ◽  
Author(s):  
Sounak Ghosh-Roy ◽  
Dhiman Das ◽  
Debarati Chowdhury ◽  
Michael J.Smerdon ◽  
Ronita Nag Chaudhuri
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Irradiation ◽  
Repair Factor ◽  
Transcription Coupled Repair

scholarly journals Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes.

1997 ◽  
Vol 17 (1) ◽  
pp. 219-229 ◽  
Author(s):  
R Dammann ◽  
G P Pfeifer
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Fibroblasts ◽  
Rna Polymerase Iii ◽  
Dna Lesions ◽  
Rrna Genes ◽  
Trna Genes ◽  
Polymerase Iii ◽  
Flanking Sequences

UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.

scholarly journals Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

1996 ◽  
Vol 16 (12) ◽  
pp. 6783-6793 ◽  
Author(s):  
S Lauder ◽  
M Bankmann ◽  
S N Guzder ◽  
P Sung ◽  
L Prakash ◽  
...  
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Excision Repair ◽  
Regulatory Element ◽  
Pol Ii ◽  
Cellular Processes ◽  
Nucleotide Excision ◽  
Damaged Dna

Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected.

scholarly journals Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 381
Author(s):  
Danielle P. Johnson ◽  
Mahesh B. Chandrasekharan ◽  
Marie Dutreix ◽  
Srividya Bhaskara
Keyword(s):  
Dna Repair ◽  
Cancer Therapy ◽  
Therapeutic Intervention ◽  
Synthetic Lethality ◽  
Checkpoint Proteins ◽  
Cellular Processes ◽  
Repair Pathways ◽  
Made In

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

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