title: Chemical and Enzymatic Footprint Analyses of R-Loop Formation by Cascade-crRNA Complex.
abstract: Cascade-crRNA complexes mediate the identification of the invading foreign DNA and initiate its neutralization by formation of an R-loop (RNA-induced DNA-loop) at the crRNA-complementary sequence (protospacer). After initial unspecific binding to the double-stranded DNA, Cascade-crRNA complex slides along the DNA to find the protospacer. Once the target site is detected, the crRNA hybridizes to the complementary strand with subsequent displacement of the non-complementary strand to form an R-loop structure. Here, we describe how Cascade-DNA complexes and the Cascade-induced strand separation can be characterized in detail by combining chemical and enzymatic footprint analyses. Selective modification of unpaired thymines by permanganate (KMnO4) and the specific cleavage of single-stranded DNA by Nuclease P1 can be used to probe an R-loop formation by Cascade. Localization of the Cascade-crRNA complex on the DNA can be achieved by an Exonuclease III protection assay.
authors.affiliation: B.R.A.I.N AG, Darmstaedter Str. 34-36, 64673, Zwingenberg, Germany, up@brain-biotech.de.
authors.forename: Ümit
authors.initials: Ü
authors.lastname: Pul
mesh_terms: D001665:Binding Sites; D053837:DNA Cleavage / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate / Q000494:pharmacology; D012313:RNA / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D011196:Potassium Permanganate; D012313:RNA; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III