title: Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.
abstract: During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Musharova
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Klimuk
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Kirill A
authors.initials: KA
authors.lastname: Datsenko
authors.affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Metlitskaya
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Logacheva
authors.affiliation: Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Semenova
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.|Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Savitskaya
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C557650:YgbT protein, E coli; D004265:DNA Helicases; C574631:ygcB protein, E coli