TY - JOUR
T1 - Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy
AU - Benureau, Yann
AU - Moreira Tavares, Eliana
AU - Muhammad, Ali Akbar
AU - Baconnais, Sonia
AU - Le Cam, Eric
AU - Dupaigne, Pauline
N1 - Publisher Copyright:
© 2020 The Author(s). Published by Oxford University Press.
PY - 2021/1/1
Y1 - 2021/1/1
N2 - 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 ammonium chloride) 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.
AB - 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 ammonium chloride) 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.
KW - DNA
KW - homologous recombination
KW - recombination intermediate
KW - replication fork
KW - replication intermediate
KW - transmission electron microscopy
UR - http://www.scopus.com/inward/record.url?scp=85100109869&partnerID=8YFLogxK
U2 - 10.1093/biomethods/bpaa012
DO - 10.1093/biomethods/bpaa012
M3 - Article
AN - SCOPUS:85100109869
SN - 2396-8923
VL - 5
SP - 1
EP - 15
JO - Biology Methods and Protocols
JF - Biology Methods and Protocols
IS - 1
ER -