Foreign DNA capture during CRISPR-Cas adaptive immunity.
Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
James K
JK
Nuñez
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Lucas B
LB
Harrington
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.|Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA.
Philip J
PJ
Kranzusch
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.|Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Alan N
AN
Engelman
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.|Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA.|Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.|Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California 94720, USA.|Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Jennifer A
JA
Doudna
D056704:Adaptive Immunity; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000821:virology; D019426:Integrases / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016662:Virus Integration / Q000235:genetics / Q000276:immunology
D064130:CRISPR-Associated Proteins; D004279:DNA, Viral; D019426:Integrases