title: Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems.
abstract: Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jiuyu
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jiazhi
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Hongtu
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Sheng
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Maolu
authors.initials: M
authors.lastname: Yin
authors.affiliation: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: ylwang@ibp.ac.cn.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; C000591054:Cas2 protein, E coli; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C557650:YgbT protein, E coli