Genome-wide distribution of Rad26 and Rad1-Rad10 reveals their relationship with Mediator and RNA polymerase II

Transcription is coupled with DNA repair, especially within nucleotide excision repair (NER). Mediator is a conserved coregulator playing a key role in RNA polymerase (Pol) II transcription. Mediator also links transcription and NER via a direct contact with Rad2/XPG endonuclease. In this work, we analyzed the genome-wide distribution of Rad26/CSB and that of Rad1-Rad10/XPF-ERCC1, addressing the question on a potential interplay of these proteins with Mediator and Pol II in yeast Saccharomyces cerevisiae. Our genome-wide analyses show that Rad1-Rad10 and Rad26 are present on the yeast genome in the absence of genotoxic stress, especially on highly transcribed regions, with Rad26 binding strongly correlating with that of Pol II. Moreover, we revealed that Rad1-Rad10 and Rad26 colocalize with Mediator on intergenic regions and physically interact with this complex. Using kin28 TFIIH mutant, we showed that Mediator stabilization on core promoters lead to an increase in Rad1-Rad10 chromatin binding, whereas Rad26 occupancy is less impacted by Mediator and follows mainly a decrease in Pol II transcription. Combined with multivariate analyses, our results reveal the interplay between Rad1-Rad10, Rad26, Mediator and Pol II, modulated by the binding dynamics of Mediator and Pol II transcription. In conclusion, we extend the Mediator link to Rad1-Rad10 and Rad26 NER proteins and reveal important differences in Mediator relationships with Rad2, Rad1-Rad10 and Rad26. Our work thus contributes to new concepts of the functional interplay between transcription and DNA repair, relevant for human diseases including cancer and XP/CS syndromes.

Download Full-text

Functional interplay between Mediator and RNA polymerase II in Rad2/XPG loading to the chromatin

Nucleic Acids Research ◽

10.1093/nar/gkz598 ◽

2019 ◽

Vol 47 (17) ◽

pp. 8988-9004 ◽

Cited By ~ 3

Author(s):

Adrien Georges ◽

Diyavarshini Gopaul ◽

Cyril Denby Wilkes ◽

Nathalie Giordanengo Aiach ◽

Elizaveta Novikova ◽

...

Keyword(s):

Dna Repair ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Excision Repair ◽

Cellular Functions ◽

Dynamic Interactions ◽

Pol Ii ◽

Core Promoters ◽

Functional Dynamics ◽

Genome Wide

Abstract Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6783 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6783-6793 ◽

Cited By ~ 43

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.00368-21 ◽

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.

Download Full-text

Mechanism of RNA polymerase II stalling by DNA alkylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706592114 ◽

2017 ◽

Vol 114 (46) ◽

pp. 12172-12177 ◽

Cited By ~ 8

Author(s):

Stefano Malvezzi ◽

Lucas Farnung ◽

Claudia M. N. Aloisi ◽

Todor Angelov ◽

Patrick Cramer ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Anticancer Agents ◽

Excision Repair ◽

Minor Groove ◽

Dna Alkylation ◽

Drug Induced ◽

Rna Pol Ii ◽

Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

Download Full-text

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Molecular and Cellular Biology ◽

10.1128/mcb.01993-06 ◽

2007 ◽

Vol 27 (5) ◽

pp. 1631-1648 ◽

Cited By ~ 104

Author(s):

Igor Chernukhin ◽

Shaharum Shamsuddin ◽

Sung Yun Kang ◽

Rosita Bergström ◽

Yoo-Wook Kwon ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Activation ◽

Ctcf Binding ◽

Pol Ii ◽

Genome Wide ◽

Target Sites ◽

On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

Download Full-text

The telomeric Cdc13–Stn1–Ten1 complex regulates RNA polymerase II transcription

Nucleic Acids Research ◽

10.1093/nar/gkz279 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6250-6268 ◽

Cited By ~ 2

Author(s):

Olga Calvo ◽

Nathalie Grandin ◽

Antonio Jordán-Pla ◽

Esperanza Miñambres ◽

Noelia González-Polo ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Elongation Factor ◽

Yeast Saccharomyces Cerevisiae ◽

Replication Fork Progression ◽

Genome Wide ◽

Length Regulation ◽

Transcription Regulators ◽

Rna Polymerase Ii Transcription

Abstract Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

Download Full-text

Nucleotide excision repair in the yeast Saccharomyces cerevisiae : its relationship to specialized mitotic recombination and RNA polymerase II basal transcription

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0010 ◽

1995 ◽

Vol 347 (1319) ◽

pp. 63-68 ◽

Cited By ~ 27

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Gene Products ◽

Basal Transcription ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleotide Excision ◽

Yeast Genes

Nucleotide excision repair (ner) in eukaryotes is a biochemically complex process involving multiple gene products. The budding yeast Saccharomyces cerevisiae is an informative model for this process. Multiple genes and in some cases gene products that are indispensable for ner have been isolated from this organism. Homologues of many of these yeast genes are structurally and functionally conserved in higher organisms, including humans. The yeast Rad1/Rad10 heterodimeric protein complex is an endonuclease that is believed to participate in damage-specific incision of DNA during ner . This endonuclease is also required for specialized types of recombination. The products of the RAD3, SSL2(RAD25) SSL1 and TFB1 genes have dual roles in ner and in RNA polymerase II-dependent basal transcription.

Download Full-text

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽

10.7554/elife.55667 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Anand Ranjan ◽

Vu Q Nguyen ◽

Sheng Liu ◽

Jan Wisniewski ◽

Jee Min Kim ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Pol Ii ◽

Promoter Escape ◽

Genome Wide ◽

A Genome ◽

Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

Download Full-text

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803275115 ◽

2018 ◽

Vol 115 (30) ◽

pp. 7777-7782 ◽

Cited By ~ 9

Author(s):

Hemanth Tummala ◽

Arran D. Dokal ◽

Amanda Walne ◽

Alicia Ellison ◽

Shirleny Cardoso ◽

...

Keyword(s):

Bone Marrow ◽

Dna Repair ◽

Rna Polymerase Ii ◽

Nuclear Dna ◽

Genome Instability ◽

Excision Repair ◽

Bone Marrow Failure ◽

Rna Pol Ii ◽

Pol Ii ◽

Repair Defect

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Download Full-text

The yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2288 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2288-2293 ◽

Cited By ~ 54

Author(s):

Z Wang ◽

S Buratowski ◽

J Q Svejstrup ◽

W J Feaver ◽

X Wu ◽

...

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleotide Excision ◽

Rna Polymerase Ii Transcription

The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal transcription and NER. Our results also suggest that the repair and transcription functions of Tfb1 and Ssl1 are separable.

Download Full-text