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

Abstract

In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, RNA polymerases stalled or paused by lesions signal the presence of DNA damage in transcriptioncoupled nucleotide excision repair (TCR). 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 damage recognition by UvrA complexes formed during GGR and TCR. 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 repair are similar in live cells, and are governed by UvrB. Here, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells. Across the various domains of life, the recognition and repair of bulky helix distorting lesions in chromosomal DNA is coordinated by nucleotide excision repair (NER) factors. Damage detection occurs in two stages: a dedicated set of damage surveillance enzymes (reviewed in ref. 1, 2, 3 ) (namely the prokaryotic UvrA, and the eukaryotic UV-DDB, XPC, XPA and homologs) constantly survey genomic DNA for lesions. Upon DNA damage recognition, these enzymes load specific factors (UvrB in prokaryotes, TFIIH and homologs in eukaryotes) that unwind the DNA and verify the location of the damage with nucleotide resolution (Fig. 1a) (reviewed in ref. 2, 3 ). Subsequently, specialized endonucleases (prokaryotic UvrC and homologs, and the eukaryotic XPF/XPG and homologs) are recruited to the site of the DNA, resulting in cleavage of the single-stranded DNA (ssDNA) patch containing the lesion (reviewed in ref. 2, 3 ).In all studied organisms, the recognition of DNA damage also occurs via the stalling of RNA polymerase at sites of lesions (reviewed in ref. 4 ). In this case, a transcription elongation complex that is unable to catalyse RNA primer extension manifests as an ultra-stable protein-DNA roadblock. Transcriptionrepair coupling factors such as the prokaryotic Mfd, and the eukaryotic homologs Rad26/CSB are dedicated factors that recognize these TECs and remodel them 5,6,7,8,9 . In prokaryotes, Mfd is recruited to the site of a failed TEC, and in turn it recruits the UvrA(B) protein (Fig. 1) 7,9,10,11 . Similarly, in eukaryotes, CSB is recruited to the site of a stalled RNAPII complex, and recruits the TFIIH complex 12 .Damage detection via elongating RNA polymerase is termed transcription-coupled repair (TCR), in contrast to the direct detection of lesions by the UvrAB damage sensor (global genomic repair, or GGR). Studies investigating the rate of repair during TCR vs. GGR, have reported an enhancement in the rate of removal of UV-induced lesions from the template strand in transcribed DNA compared to 14...

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

“…Further, the lifetime of 143 s closely matches that of UvrA-YPet in uvrA-YPet uvrB cells (97 ± 18 s) 21 . Taken together, these results indicate that a highly stable DNA-bound Mfd-UvrA2 complex is formed in the absence of UvrB (Fig.…”

“…4a-b), followed by interrogation of the nucleobases mediated by UvrB's cryptic helicase activity 29,30 . Since loading of UvrB promotes dissociation of UvrA from the damage surveillance complex 21 , we next investigated whether dissociation of Mfd from the handoff complex occurs upon loading of UvrB. We measured the residence time of Mfd-YPet in cells expressing β-hairpin mutants of UvrB from the native chromosomal locus 27,29,31 (Extended Data Fig.…”

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

“…n bacteria, transcription and DNA replication occur concomitantly, making potentially damaging collisions of DNA replication forks with transcription complexes inevitable [1][2][3][4][5] . Transcription is highly sensitive to DNA damage, which causes the elongation complex (EC) to pause, and multiple redundant systems have evolved to ensure rapid removal of RNAP and/or the repair of damaged DNA [6][7][8][9][10] . This reduces the chance of replication forks colliding with stalled transcription complexes whilst also serving as an efficient system for maintaining genome integrity, especially within coding regions.…”

Mechanism of transcription modulation by the transcription-repair coupling factor

“…A detailed understanding of the activities of Mfd has been assembled through a combination of molecular biology, biochemical (Chambers et al, 2003;Haines et al, 2014;Manelyte et al, 2010;Park et al, 2002;Selby and Sancar, 1993a, b;Smith et al, 2012;Smith et al, 2007), structural methods (Brugger et al, 2020;Deaconescu et al, 2006;Kang et al, 2021;Shi et al, 2020), as well as singlemolecule (Fan et al, 2016;Graves et al, 2015;Howan et al, 2012;Le et al, 2017) and live-cell imaging techniques (Ghodke et al, 2020;Ho et al, 2018Ho et al, , 2020. Mfd acts in a complex role in modulating transcription activity, including at hard-to-transcribe regions (Ragheb et al, 2021).…”