A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
02 12 2021
02 12 2021
Historique:
accepted:
26
08
2021
revised:
15
08
2021
received:
05
03
2021
pubmed:
10
9
2021
medline:
11
1
2022
entrez:
9
9
2021
Statut:
ppublish
Résumé
We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.
Identifiants
pubmed: 34500456
pii: 6368053
doi: 10.1093/nar/gkab770
pmc: PMC8643652
doi:
Substances chimiques
DNA, Mitochondrial
0
DNA, Single-Stranded
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e121Informations de copyright
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
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