title: Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.
abstract: CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Huo
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2] [3].
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Nam
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
authors.forename: Fang
authors.initials: F
authors.lastname: Ding
authors.affiliation: 1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
authors.forename: Heejin
authors.initials: H
authors.lastname: Lee
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai, China.
authors.forename: Lijie
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Yibei
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Farchione
authors.affiliation: Howard Hughes Medical Institute, Mass Spectrometry Laboratory, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Sharleen
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Northeastern Collaborative Access Team and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, Illinois, USA.
authors.forename: Kanagalaghatta
authors.initials: K
authors.lastname: Rajashankar
authors.affiliation: Northeastern Collaborative Access Team and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, Illinois, USA.
authors.forename: Igor
authors.initials: I
authors.lastname: Kurinov
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai, China.
authors.forename: Rongguang
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Ailong
authors.initials: A
authors.lastname: Ke
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D064130:CRISPR-Associated Proteins; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004265:DNA Helicases