Biology subjects

Lepore, A.

Publications and source records attributed to Lepore, A..

4 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↗

bioRxiv2021

Previous SARS-CoV-2 infection increases B.1.1.7 cross-neutralization by vaccinated individuals.

To assess the potential impact of predominant circulating SARS-CoV-2 variants on neutralizing activity of infected and/or vaccinated individuals, we analyzed neutralization of pseudoviruses expressing the spike of the original Wuhan strain, the D614G and B.1.1.7 variants. Our data show that parameters of natural infection (time from infection and infecting variant) determined cross-neutralization. Importantly, upon vaccination, previously infected individuals developed equivalent B.1.1.7 and Wuhan neutralizing responses. In contrast, uninfected vaccinees showed reduced neutralization against B.1.1.7. FundingThis study was funded by Grifols, the Departament de Salut of the Generalitat de Catalunya, the Spanish Health Institute Carlos III, CERCA Programme/Generalitat de Catalunya, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos.

immunology↗

bioRxiv2017

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is [≥]80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

systems biology↗