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

Single Molecule ◽

Excision Repair ◽

Model Organism ◽

Coupling Factor ◽

Live Cells ◽

Nucleotide Excision ◽

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.

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair

10.1101/515502 ◽

2019 ◽

Cited By ~ 1

Author(s):

Harshad Ghodke ◽

Han Ngoc Ho ◽

Antoine M van Oijen

Keyword(s):

Single Molecule ◽

Excision Repair ◽

Model Organism ◽

Live Cells ◽

Exogenous Dna ◽

Damage Recognition ◽

Nucleotide Excision ◽

Nucleotide Excision Repair Pathway ◽

Kinetics Of

AbstractIn the model organismEscherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled or paused by lesions. Ultimately, damage recognition is mediated by UvrA, culminating in the loading of the damage verification enzyme UvrB. We set out to characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB. We followed functional, fluorescently tagged UvrA molecules in live cells and measured their residence times in TCR-deficient or wild-type cells. We demonstrate that the lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, we found that the lifetimes of UvrA strongly depended on, and matched those of Mfd. Here, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.

A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro

Mutation Research/DNA Repair ◽

10.1016/0921-8777(96)00019-5 ◽

1996 ◽

Vol 364 (1) ◽

pp. 33-41 ◽

Cited By ~ 13

Author(s):

Zhigang Wang ◽

Xiaohua Wu ◽

Errol C. Friedberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Excision Repair ◽

Cell Extract ◽

Nucleotide Excision ◽

Transcription In Vitro ◽

Rna Polymerase Ii Transcription ◽

Whole Cell Extract

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716581115 ◽

2018 ◽

Vol 115 (29) ◽

pp. E6770-E6779 ◽

Cited By ~ 6

Author(s):

Laurianne Daniel ◽

Elena Cerutti ◽

Lise-Marie Donnio ◽

Julie Nonnekens ◽

Christophe Carrat ◽

...

Keyword(s):

Rna Polymerase ◽

Uv Irradiation ◽

Ribosomal Dna ◽

Excision Repair ◽

Rna Polymerase I ◽

Nucleotide Excision ◽

Polymerase I ◽

Uv Lesions ◽

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.

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 ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

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.

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 ◽

Bound States ◽

Excision Repair ◽

Structural Basis ◽

Template Strand ◽

Cryo Electron Microscopy ◽

Nucleotide Excision ◽

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.

Yeast RNA Polymerase II Transcription In Vitro Is Inhibited in the Presence of Nucleotide Excision Repair: Complementation of Inhibition by Holo-TFIIH and Requirement for RAD26

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2668 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2668-2676 ◽

Cited By ~ 29

Author(s):

Zhaoyang You ◽

William J. Feaver ◽

Errol C. Friedberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Excision Repair ◽

Cell Extracts ◽

Nucleotide Excision ◽

Rnap Ii ◽

Group B ◽

Damaged Dna

ABSTRACT The Saccharomyces cerevisiae transcription factor IIH (TFIIH) is essential both for transcription by RNA polymerase II (RNAP II) and for nucleotide excision repair (NER) of damaged DNA. We have established cell extracts which support RNAP II transcription from the yeast CYC1 promoter or NER of transcriptionally silent damaged DNA on independent plasmid templates and substrates. When plasmid templates and substrates for both processes are simultaneously incubated with these extracts, transcription is significantly inhibited. This inhibition is strictly dependent on active NER and can be complemented with purified holo-TFIIH. These results suggest that in the presence of active NER, TFIIH is preferentially mobilized from the basal transcription machinery for use in NER. Inhibition of transcription in the presence of active NER requires theRAD26 gene, the yeast homolog of the human Cockayne syndrome group B gene (CSB).

Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3041-3049.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3041-3049

Author(s):

L Bardwell ◽

A J Cooper ◽

E C Friedberg

Keyword(s):

Dna Repair ◽

Excision Repair ◽

Specific Interaction ◽

Mitotic Recombination ◽

Nucleotide Excision ◽

Specific Association ◽

Recombination Pathway ◽

Cloned Gene

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.

Nucleotide Excision Repair in Human Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m113.482257 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20918-20926 ◽

Cited By ~ 52

Author(s):

Jinchuan Hu ◽

Jun-Hyuk Choi ◽

Shobhan Gaddameedhi ◽

Michael G. Kemp ◽

Joyce T. Reardon ◽

...

Keyword(s):

Genomic Dna ◽

Excision Repair ◽

Human Cells ◽

Naked Dna ◽

Nucleotide Excision ◽

Uv Photoproducts ◽

Mutant Cells

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.