1 research outputs found
Lighting Up Individual DNA Damage Sites by In Vitro Repair Synthesis
DNA
damage and repair are linked to fundamental biological processes
such as metabolism, disease, and aging. Single-strand lesions are
the most abundant form of DNA damage; however, methods for characterizing
these damage lesions are lacking. To avoid double-strand breaks and
genomic instability, DNA damage is constantly repaired by efficient
enzymatic machinery. We take advantage of this natural process and
harness the repair capacity of a bacterial enzymatic cocktail to repair
damaged DNA in vitro and incorporate fluorescent nucleotides into
damage sites as part of the repair process. We use single-molecule
imaging to detect individual damage sites in genomic DNA samples.
When the labeled DNA is extended on a microscope slide, damage sites
are visualized as fluorescent spots along the DNA contour, and the
extent of damage is easily quantified. We demonstrate the ability
to quantitatively follow the damage dose response to different damaging
agents as well as repair dynamics in response to UV irradiation in
several cell types. Finally, we show the modularity of this single-molecule
approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine
in genomic DNA extracted from mouse brain tissue