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

2019 ◽  
Author(s):  
Harshad Ghodke ◽  
Han Ngoc Ho ◽  
Antoine M van Oijen
Keyword(s):  
Nucleotide Excision Repair ◽  
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.

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

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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