Recombinant Escherichia coli CRISPR-associated endoribonuclease Cas2 (ygbF)

CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). The Cas1-Cas2 complex is involved in CRISPR adaptation, the first stage of CRISPR immunity, being required for the addition/removal of CRISPR spacers at the leader end of the CRISPR locus. The Cas1-Cas2 complex introduces staggered nicks into both strands of the CRISPR array near the leader repeat and joins the 5'-ends of the repeat strands with the 3'-ends of the new spacer sequence. Spacer DNA integration requires supercoiled target DNA and 3'-OH ends on the inserted (spacer) DNA and probably initiates with a nucleophilic attack of the C 3'-OH end of the protospacer on the minus strand of the first repeat sequence. Expression of Cas1-Cas2 in a strain lacking both genes permits spacer acquisition. Cas2 not seen to bind DNA alone; the Cas1-Cas2 complex preferentially binds CRISPR-locus DNA. Highest binding is seen to a dual forked DNA complex with 3'-overhangs and a protospacer-adjacent motif-complement specifically positioned. The protospacer DNA lies across a flat surface extending from 1 Cas1 dimer, across the Cas2 dimer and contacting the other Cas1 dimer; the 23 bp-long ds section of the DNA is bracketed by 1 Tyr-22 from each of the Cas1 dimers. Cas1 cuts within the 3'-overhang, to generate a 33-nucleotide DNA that is probably incorporated into the CRISPR leader by a cut-and-paste mechanism. This subunit's probable nuclease activity is not required for spacer acquisition.