Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems
DOI： http://dx.doi.org/10.1016/j.cell.2015.10.008Bacteria
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.
本文选自：生物谷
