Foreign DNA capture during CRISPR–Cas adaptive immunity
Nuñez, James K.
Harrington, Lucas B.
Kranzusch, Philip J.
Doudna, Jennifer A.
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CitationNuñez, James K., Lucas B. Harrington, Philip J. Kranzusch, Alan N. Engelman, and Jennifer A. Doudna. 2015. “Foreign DNA capture during CRISPR–Cas adaptive immunity.” Nature 527 (7579): 535-538. doi:10.1038/nature15760. http://dx.doi.org/10.1038/nature15760.
AbstractBacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. 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 (nt) 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 vivo2–4. 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.
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