title: Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system.
abstract: The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Zihni
authors.initials: Z
authors.lastname: Arslan
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Veronica
authors.initials: V
authors.lastname: Hermanns
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Reinhild
authors.initials: R
authors.lastname: Wurm
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Wagner
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany pul@hhu.de.
authors.forename: Ümit
authors.initials: Ü
authors.lastname: Pul
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D004247:DNA; D003851:Deoxyribonucleases