title: Lighting up individual DNA damage sites by in vitro repair synthesis.
abstract: 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.
authors.affiliation: Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University , Max and Anna Web Street, Ramat-Gan, Israel 5290002.
authors.forename: Shahar
authors.initials: S
authors.lastname: Zirkin
authors.forename: Sivan
authors.initials: S
authors.lastname: Fishman
authors.forename: Hila
authors.initials: H
authors.lastname: Sharim
authors.forename: Yael
authors.initials: Y
authors.lastname: Michaeli
authors.forename: Jeremy
authors.initials: J
authors.lastname: Don
authors.forename: Yuval
authors.initials: Y
authors.lastname: Ebenstein
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004260:DNA Repair; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D051760:Xeroderma Pigmentosum Group A Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA