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

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.

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal‐Free Electro‐Stain Composed of DNA‐Binding Proteins and Synthetic Polymers

“…[ 23 ] The thickness of the metal casting is typically ≈8 nm for high‐contrast TEM images. [ 24 ] AFM studies have demonstrated that the diameter of DNA ranges between 5 and 10 nm, although they have also revealed a sub‐nanometer helical groove pattern within the DNA backbone. [ 25 ] Furthermore, several studies utilizing super‐resolution microscopy methods have been reported, with the finest resolution for DNA imaging being 45 nm using STED (Stimulated Emission Depletion), [ 8a ] 14 nm using BALM (Binding Activated Localization Microscopy), [ 8b ] 67 nm using STORM (Stochastic Optical Reconstruction Microscopy), [ 26 ] and 20 nm using Vortex PSF (Point Spread Function).…”

A transcriptional mechanism involving R-loop, m6A modification and RNA abasic sites regulates an enhancer RNA of APOE

“…Here, we asked whether MPG affects APOE expression through binding to R-loops. Using the S9.6 antibody that specifically recognizes R-loops (Hu et al, 2006; Kosar et al, 2021), we carried out DNA-RNA immunoprecipitation, DRIP, followed by sequencing. Since APOE is not transcribed or at a very low level in fibroblasts, there was no RNA to form R-loops in APOE , rather we detected an R-loop upstream of APOE (Figure 2A, top panel), in an intergenic region.…”

Toward Visualizing Genomic DNA Using Electron Microscopy via DNA Metallization

“…In addition to the study of genomic DNA, EM has also been used to characterize the mitochondrial DNA replication cycle, as shown in Figure 1F. [41] Furthermore, DNA EM allows for visualization of various structures such as plasmid DNA, ss-ds DNA, ssDNA gap between dsDNA, holiday junction of DNA, D-loop, strand exchanges, and DNA intermediates formed during DNA damage repair. [33,42] 2.2.…”

“…F) Mitochondrial D-loops and some single-stranded DNA denaturation bubbles via uranyl acetate and Pt shadowing. Reproduced with permission [41]. Copyright 2021, Oxford University Press.…”