Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy

Yann Benureau (1, 2) , Eliana Moreira Tavares , Ali-Akbar Muhammad , Sonia Baconnais (3, 1, 4) , Eric Le Cam (3, 1, 4) , Pauline Dupaigne (5)

1 IGR - Institut Gustave Roussy
2 IGC - Intégrité du génome et cancers
3 CNRS - Centre National de la Recherche Scientifique
4 UMR8126 - Signalisation, noyaux et innovations en cancérologie
5 INSERM - Institut National de la Santé et de la Recherche Médicale

DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes. These particular DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl alkyl ammoniumchloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA intermediates.

Mots clés: Electron Microscopy; DNA replication and DNA repair; transmission electron microscopy; DNA; replication fork; homologous recombination; replication intermediate; recombination intermediate

Domaines: Sciences du Vivant [q-bio]

Yann Benureau, Eliana Moreira Tavares, Ali-Akbar Muhammad, Sonia Baconnais, Eric Le Cam, et al.. Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy. Biology Methods and Protocols, 2020, 5 (1), ⟨10.1093/biomethods/bpaa012⟩. ⟨hal-03036224⟩