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Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy


Stoy, Henriette; Luethi, Joel; Roessler, Fabienne K; Riemann, Johannes; Kaech, Andres; Lopes, Massimo (2022). Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy. In: Aguilera, Andrés; Ruzov, Alexey. R-Loops: Methods and Protocols. New York: Springer, 1-20.

Abstract

R-loop are physiologically present on genomic DNA of different organisms and play important roles in genome regulation. However, an increase in their abundance and/or size has been suggested to interfere with the DNA replication process, contributing to genome instability. Most available approaches to monitor R-loops are based on antibodies/enzymes that cannot effectively distinguish R-loops from DNA-RNA hybrids and assess R-loop size and frequency in a population of molecules. Electron microscopy has successfully allowed single-molecule visualization of DNA replication and repair intermediates, uncovering key architectural modifications in DNA, induced by genotoxic stress or by the associated cellular response. Here, we describe recent modifications of this visualization workflow to implement partial automation of image acquisition and analysis. Coupling this refined workflow with sample preparation procedures that protect R-loop stability allows for direct visualization of R-loop structures on genomic DNA, independently from probes. Combining single-molecule information and DNA content assessment, this approach provides direct estimations of R-loop frequency, size, and burden on genomic DNA.

Abstract

R-loop are physiologically present on genomic DNA of different organisms and play important roles in genome regulation. However, an increase in their abundance and/or size has been suggested to interfere with the DNA replication process, contributing to genome instability. Most available approaches to monitor R-loops are based on antibodies/enzymes that cannot effectively distinguish R-loops from DNA-RNA hybrids and assess R-loop size and frequency in a population of molecules. Electron microscopy has successfully allowed single-molecule visualization of DNA replication and repair intermediates, uncovering key architectural modifications in DNA, induced by genotoxic stress or by the associated cellular response. Here, we describe recent modifications of this visualization workflow to implement partial automation of image acquisition and analysis. Coupling this refined workflow with sample preparation procedures that protect R-loop stability allows for direct visualization of R-loop structures on genomic DNA, independently from probes. Combining single-molecule information and DNA content assessment, this approach provides direct estimations of R-loop frequency, size, and burden on genomic DNA.

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research

04 Faculty of Medicine > Center for Microscopy and Image Analysis
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:2022
Deposited On:16 Mar 2023 09:18
Last Modified:28 Mar 2024 04:40
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:2528
ISSN:1064-3745
ISBN:9781071624760
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-0716-2477-7_1
PubMed ID:35704181