Biology subjects

Thedie, D.

Publications and source records attributed to Thedie, D..

5 recordsLinked to original sources

bioRxiv2025

Single molecule imaging of RecB in vivo reveals dynamics of DNA Double Strand Break repair in Escherichia coli

Repairing DNA damage is of primary importance for all living organisms. DNA double-strand breaks (DSBs) are one of the most serious types of DNA damage, as they lead to loss of genetic information and death when not repaired. In E. coli, they are recognised and processed by the RecBCD complex, which initiates repair by homologous recombination. Although the repair dynamics down-stream of RecBCD has been well characterised, it is still unclear how long this complex stays attached to DNA and what triggers its dissociation in vivo. To answer these questions, we imaged RecB at the single-molecule level, and quantified its dynamic behaviour in bacterial cells exposed to ciprofloxacin, an antibiotic that induces DSBs. Our results show that RecB forms long-lived complexes with DSBs (10 seconds), and that its dissociation from DNA is an intrinsic property of the complex, that does not depend on the amount of DNA damage, nor the following steps in the repair pathway. Moreover, we show that we can use RecB binding to DSBs as a marker to estimate the rate of damage formation. This study provides a detailed quantitative insight into the interaction of RecBCD with DNA double-strand ends in E. coli in vivo, and into the bacterial response to DSBs induced by ciprofloxacin.

microbiology↗

bioRxiv2023

In vivo single-molecule imaging of RecB reveals efficient repair of DNA damage in Escherichia coli

Efficient DNA repair is crucial for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends following a DNA double-strand break (DSB) to initiate repair by homologous recombination. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we perform single-molecule microscopy to investigate DNA repair in real-time in E. coli. We quantify RecB single-molecule mobility and monitor the induction of the DNA damage response (SOS response) in individual cells. We show that RecB binding to broken DNA ends leads to efficient repair without SOS induction. In contrast, in a RecB mutant with modified activities leading to the activation of an alternative repair pathway, repair is less efficient and leads to high SOS induction. Our findings reveal how subtle alterations in RecB activity profoundly impact the efficiency of DNA repair in E. coli.

molecular biology↗

bioRxiv2022

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at [~]110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/504779v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1d5ed02org.highwire.dtl.DTLVardef@1bb2359org.highwire.dtl.DTLVardef@10dc6b1org.highwire.dtl.DTLVardef@16fcd94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state. TOC GRAPHICS O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2018

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort.

biophysics↗