Mechanistic insights in transcription-coupled nucleotide excision repair of ribosomal DNA

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.

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Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand- specific nucleotide excision repair of RNA polymerase I transcribed genes

Nucleic Acids Research ◽

10.1093/nar/24.6.1020 ◽

1996 ◽

Vol 24 (6) ◽

pp. 1020-1025 ◽

Cited By ~ 28

Author(s):

R. Verhage

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Rna Polymerase I ◽

Nucleotide Excision ◽

Polymerase I ◽

Specific Nucleotide

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RNA Polymerase-I-Dependent Transcription-coupled Nucleotide Excision Repair of UV-Induced DNA Lesions at Transcription Termination Sites, inSaccharomyces cerevisiae

Photochemistry and Photobiology ◽

10.1111/php.12690 ◽

2017 ◽

Vol 93 (1) ◽

pp. 363-374 ◽

Cited By ~ 1

Author(s):

François Peyresaubes ◽

Carlos Zeledon ◽

Laetitia Guintini ◽

Romain Charton ◽

Alexia Muguet ◽

...

Keyword(s):

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Transcription Termination ◽

Dna Lesions ◽

Rna Polymerase I ◽

Nucleotide Excision ◽

Polymerase I

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Single-molecule imaging reveals molecular coupling between transcription and DNA repair in live cells

10.1101/515536 ◽

2019 ◽

Author(s):

Han Ngoc Ho ◽

Antoine van Oijen ◽

Harshad Ghodke

Keyword(s):

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Single Molecule ◽

Excision Repair ◽

Model Organism ◽

Coupling Factor ◽

Live Cells ◽

Nucleotide Excision ◽

Transcription Coupled Repair

Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair1–3. During this reaction, stalled transcription elongation complexes at sites of lesions serve as a signal to trigger the assembly of nucleotide excision repair factors (reviewed in ref.4,5). In the model organism Escherichia coli, the transcription-repair coupling factor Mfd displaces the stalled RNA polymerase and hands-off the stall site to the nucleotide excision repair factors UvrAB for damage detection6–9. Despite in vitro evidence, it remains unclear how in live cells the stall site is faithfully handed over to UvrB from RNA polymerase and whether this handoff occurs via the Mfd-UvrA2-UvrB complex or via alternate reaction intermediates. Here, we visualise Mfd, the central player of transcription-coupled repair in actively growing cells and determine the catalytic requirements for faithful completion of the handoff during transcription-coupled repair. We find that the Mfd-UvrA2 complex is arrested on DNA in the absence of UvrB. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition, were also impaired in successful handoff. Our observations demonstrate that in live cells, the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner during transcription-coupled repair.

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Structural basis for transcription complex disruption by the Mfd translocase

eLife ◽

10.7554/elife.62117 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jin Young Kang ◽

Eliza Llewellyn ◽

James Chen ◽

Paul Dominic B Olinares ◽

Joshua Brewer ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

Transcription Coupled Repair

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

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Structural basis for transcription complex disruption by the Mfd translocase

10.1101/2020.08.12.248500 ◽

2020 ◽

Author(s):

Jin Young Kang ◽

Eliza Llewellyn ◽

James Chen ◽

Paul Dominic B. Olinares ◽

Joshua Brewer ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Nucleotide Excision Repair ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

Transcription Coupled Repair

SummaryTranscription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

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Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation

Nucleic Acids Research ◽

10.1093/nar/gkz977 ◽

2019 ◽

Cited By ~ 1

Author(s):

Frauke Liebelt ◽

Joost Schimmel ◽

Matty Verlaan – de Vries ◽

Esra Klemann ◽

Martin E van Royen ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nucleotide Excision Repair ◽

Uv Irradiation ◽

Excision Repair ◽

Premature Aging ◽

Dependent Manner ◽

Ubiquitin Ligase Complex ◽

Nucleotide Excision ◽

Damage Response

Abstract Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.

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A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation

Molecular Biology International ◽

10.1155/2014/519290 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8

Author(s):

Bernadette Connors ◽

Lauren Rochelle ◽

Asela Roberts ◽

Graham Howard

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Excision Repair ◽

Uv Irradiation ◽

Double Mutant ◽

Excision Repair ◽

Strand Break ◽

Anaphase Promoting Complex ◽

End Joining ◽

Ubiquitin Ligase Complex ◽

Nucleotide Excision

Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair.

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