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Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy


Stoy, Henriette; Lang, Kevin S; Merrikh, Houra; Lopes, Massimo (2022). Locus-Specific Analysis of Replication Dynamics and Detection of DNA-RNA Hybrids by Immuno Electron Microscopy. In: Aguilera, Andrés; Ruzov, Alexey. R-Loops: Methods and Protocols. New York: Springer, 67-89.

Abstract

DNA-RNA hybrids can interfere with DNA replication, but the underlying intermediates and molecular mechanisms have remained elusive. Here, we describe a single molecule approach that allows to monitor DNA-RNA hybrids locus-specifically in the context of ongoing replication. Using restriction digestion, gel electrophoresis and gel elution, this workflow allows to efficiently isolate replication intermediates and to study replication dynamics across a specific genomic locus. Here, we applied this procedure to isolate a bacterial genomic locus carrying an inducible transcription-replication conflict. Moreover, we combined electron microscopy with S9.6-Gold immuno-labeling to detect DNA-RNA hybrids on the isolated replication intermediates. With some limitations, this approach may be adapted to locus-specific replication analyses in different organisms.

Abstract

DNA-RNA hybrids can interfere with DNA replication, but the underlying intermediates and molecular mechanisms have remained elusive. Here, we describe a single molecule approach that allows to monitor DNA-RNA hybrids locus-specifically in the context of ongoing replication. Using restriction digestion, gel electrophoresis and gel elution, this workflow allows to efficiently isolate replication intermediates and to study replication dynamics across a specific genomic locus. Here, we applied this procedure to isolate a bacterial genomic locus carrying an inducible transcription-replication conflict. Moreover, we combined electron microscopy with S9.6-Gold immuno-labeling to detect DNA-RNA hybrids on the isolated replication intermediates. With some limitations, this approach may be adapted to locus-specific replication analyses in different organisms.

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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
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 09:41
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_6
PubMed ID:35704186