Identification of multiple kinetic populations of DNA-binding proteins in live cells

Ho, Han Ngoc; Zalami, Daniel; Köhler, Jürgen; Oijen, Antoine M. van; Ghodke, Harshad

doi:10.1101/509620

Cited by 3 publications

(6 citation statements)

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“…To estimate the influence of the number of simulated reaction events on the accuracy of the inferred rate spectra for the case of two simulated dissociation rates with variable spacing, we varied the number of simulated reaction events between 100 and 10.000 per time-lapse condition and quantified the deviation from the ground truth using 100 independent simulations (Figure 2d and Methods). In line with 23 , the closer the reaction rates, the more reaction events need to be observed to resolve them.…”

Section: Analysing Superimposed Reactions By Gridmentioning

confidence: 65%

Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

Reisser

Hettich

Kühn

et al. 2019

Preprint

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Actions of molecular species, for example binding of transcription factors to chromatin, are intrinsically stochastic and may comprise several mutually exclusive pathways. Inverse Laplace transformation in principle resolves the rate constants and frequencies of superimposed reaction processes, however current approaches are challenged by single molecule fluorescence time series prone to photobleaching. Here, we present a genuine rate identification method (GRID) that infers the quantity, rates and frequencies of dissociation processes from single molecule fluorescence survival time distributions using a dense grid of possible decay rates. In particular, GRID is able to resolve broad clusters of rate constants not accessible to common models of one to three exponential decay rates. We validate GRID by simulations and apply it to the problem of in-vivo TF-DNA dissociation, which recently gained interest due to novel single molecule imaging technologies. We consider dissociation of the transcription factor CDX2 from chromatin. GRID resolves distinct, decay rates and identifies residence time classes overlooked by other methods. We confirm that such sparsely distributed decay rates are compatible with common models of TF sliding on DNA.

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Section: Analysing Superimposed Reactions By Gridmentioning

confidence: 65%

Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

Reisser

Hettich

Kühn

et al. 2019

Preprint

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“…Consequently, measurement of interactions that last longer than the photobleaching lifetime is impossible. Instead we imaged UvrA-YPet using an interval imaging strategy 10,22,27,28 (Fig. 2d) that elegantly deconvolutes the lifetime of the interaction of UvrA-YPet with DNA and the lifetime of the fluorescent probe.…”

Section: Interval Imaging Strategy To Measure Dna Binding Kineticsmentioning

confidence: 99%

“…The effective off-rate constant keff, contributively of the photobleaching rate kb and the off-rate koff, was obtained by fitting the cumulative residence time distribution to a single-exponential model. The corresponding kefftl vs. tl plot was obtained as described previously 10,22 , with the shaded error bar representing standard deviations of ten bootstrapped samples deriving from 80% of the complied binding events (custom-written MATLAB codes) 22 . In experiments involving rifampicin treatment, cells were incubated in growth media containing rifampicin (50 μg per mL) for 30 min in the flow cell prior to imaging.…”

Section: Interval Imaging For Dissociation Kinetics Measurementsmentioning

confidence: 99%

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

Ghodke

Oijen

2019

Preprint

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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...

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“…To extend the observation time window, we adopted interval imaging in which consecutive exposure times were spaced out by the addition of a dark time (d) (see ref. 19,20 and Methods). We used a large set of distinct d covering three orders of magnitude (0.1 s to 9.9 s) to ensure accurate measurements of binding lifetimes that last for seconds to several minutes inside cells ( Fig.…”

mentioning

confidence: 99%

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

Oijen²,

Ghodke³

2019

Preprint

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Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair 1-3 . 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 detection 6-9 . 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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Identification of multiple kinetic populations of DNA-binding proteins in live cells

Cited by 3 publications

References 44 publications

Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

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

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

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