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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References
Sogo JM, Lopes M, Foiani M (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297:599–602
Lopes M, Foiani M, Sogo JM (2006) Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol Cell 21:15–27
Quinet A, Tirman S, Jackson J, Šviković S, Lemaçon D, Carvajal-Maldonado D et al (2020) PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient cells. Mol Cell 77:461–474
Berti M, Cortez D, Lopes M (2020) The plasticity of DNA replication forks in response to clinically relevant genotoxic stress. Nat Rev Mol Cell Biol 21:663–651
Kolinjivadi AM, Sannino V, De Antoni A, Zadorozhny K, Kilkenny M, Técher H et al (2017) Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Mol Cell 67:867–881
Mukherjee C, Tripathi V, Manolika EM, Heijink AM, Ricci G, Merzouk S et al (2019) RIF1 promotes replication fork protection and efficient restart to maintain genome stability. Nat Commun 10:3287
Ray Chaudhuri A, Hashimoto Y, Herrador R, Neelsen KJ, Fachinetti D, Bermejo R et al (2012) Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat Struct Mol Biol 19:417–423
Follonier C, Oehler J, Herrador R, Lopes M (2013) Friedreich’s ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions. Nat Struct Mol Biol 20:486–494
Giannattasio M, Zwicky K, Follonier C, Foiani M, Lopes M, Branzei D (2014) Visualization of recombination-mediated damage bypass by template switching. Nat Struct Mol Biol 21:884–892
Zellweger R, Dalcher D, Mutreja K, Berti M, Schmid JA, Herrador R et al (2015) Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. J Cell Biol 208:563–579
Mijic S, Zellweger R, Chappidi N, Berti M, Jacobs K, Mutreja K et al (2017) Replication fork reversal triggers fork degradation in BRCA2-defective cells. Nat Commun 8:859
Lemacon D, Jackson J, Quinet A, Brickner JR, Li S, Yazinski S et al (2017) MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat Commun 8:860
Mutreja K, Krietsch J, Hess J, Ursich S, Berti M, Roessler FK et al (2018) ATR-mediated global fork slowing and reversal assist fork traverse and prevent chromosomal breakage at DNA interstrand cross-links. Cell Rep 24:2629–2642
Zellweger R, Lopes M (2018) Dynamic architecture of eukaryotic DNA replication forks in vivo, visualized by electron microscopy. Methods Mol Biol 1672:261–294
Lopes M (2009) Electron microscopy methods for studying in vivo DNA replication intermediates. In: DNA replication. Springer, pp 605–631
Neelsen KJ, Chaudhuri AR, Follonier C, Herrador R, Lopes M (2014) Visualization and interpretation of eukaryotic DNA replication intermediates in vivo by electron microscopy. In: Functional analysis of DNA and chromatin. Springer, pp 177–208
Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465
Stoy H, Zwicky K, Lang K, Krietsch J, Kuster D, Schmid J et al Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids. under Revis. after peer Rev
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Stoy, H., Luethi, J., Roessler, F.K., Riemann, J., Kaech, A., Lopes, M. (2022). Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy. In: Aguilera, A., Ruzov, A. (eds) R-Loops . Methods in Molecular Biology, vol 2528. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2477-7_1
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DOI: https://doi.org/10.1007/978-1-0716-2477-7_1
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