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.

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy / M. Kosar, D. Piccini, M. Foiani, M. Giannattasio. - In: NUCLEIC ACIDS RESEARCH. - ISSN 1362-4962. - 49:21(2021 Dec 02), pp. e121-e139. [10.1093/nar/gkab770]

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

M. Foiani
Penultimo
Funding Acquisition
;
M. Giannattasio
Ultimo
Project Administration
2021

Abstract

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.
Mitochondrial DNA replication; Transmission Electron Microscopy; D-loop structure; Strand Displacement
Settore BIO/11 - Biologia Molecolare
2-dic-2021
https://academic.oup.com/nar/article/49/21/e121/6368053?login=true
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/987308
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