Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity - PubMed (original) (raw)

Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity

James K Nuñez et al. Nat Struct Mol Biol. 2014 Jun.

Abstract

The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.

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Figures

Figure 1

Figure 1. Cas1 and Cas2 associate to form a complex

(a) Representation of the CRISPR–Cas locus of E. coli K12. The 33-bp spacers (squares) are separated by 28-bp repeats (black diamonds). The half arrows flanking the leader and repeat-spacer arrays represent the positions of the primers used for PCR amplification in the spacer acquisition assays in BL21-AI cells. (b) Agarose gel of the PCR amplified CRISPR-I locus of BL21-AI cells after induced expression of empty vector, Cas1, Cas2 or Cas1+Cas2. Distinct bands represent the number of repeat-spacer arrays additions into the genomic parental CRISPR locus. (c) FLAG- and HA-immunoprecipitations in lysates overexpressing Cas1 only, Cas2 only or both. (d) ITC trace of Cas1 injection into a Cas2-containing cell. The reported N and _K_d values are averages of three independent experiments.

Figure 2

Figure 2. Crystal structure of the Cas1–Cas2 complex

(a) The overall structure consists of a Cas2 dimer (yellow and orange) and two Cas1 dimers (a-d, blue and teal). (b) Superposition of the Cas1a-b dimer with the previously determined E. coli Cas1 structure (gray, PDB 3NKD). The dashed orange circle highlights the conformational change observed in the a-helical domain of Cas1a. (c) Superposition of the Cas2 dimer in the complex with the previously determined E. coli Cas2 structure (gray, PDB 4MAK). The arrows point to the last resolved residue in the 4MAK structure. The N and C indicate the termini of each monomer; root-mean-square deviations (r.m.s.d.) of the superpositions are indicated.

Figure 3

Figure 3. Disruption of complex formation affects spacer acquisition in vivo

(a) A close-up view of the Cas1a–Cas2 protein-protein interface, with annotations for the residues involved in electrostatic interactions. (b) View of the ordered C-terminal tail of Cas1a with the electron density mesh contoured at 1.0 sigma. (c–e) Agarose gels of in vivo acquisition assays with mutations of Cas1 and Cas2 at the C-termini (c) and the electrostatic interface (d,e). (f) Western blot of FLAG immunoprecipitations in BL21-AI cells expressing Cas1-FLAG and Cas2-HA or various mutations of Cas1 and Cas2. Despite the low expression of Cas2 E65R, we still detect its co-elution with Cas1.

Figure 4

Figure 4. Complex formation is required for CRISPR DNA recognition

(a,b) Close-up views of the Cas1 and Cas2 active sites with stick representations for the conserved residues mutated in vivo. In the middle is a general view of the active sites in the complex, highlighted in red. (c,d) Acquisition assays of active site residue mutations of Cas1 and Cas2. (e) Western blot of fractions in the biotinylated DNA affinity precipitations. The cartoon representations are the DNA constructs used with the 5′-biotin labels (stars). W.C.L. refers to the whole cell lysate. (f) Western blot of the DNA affinity precipitations in BL21-AI lysates from overexpression of Cas1-FLAG only, Cas2-HA only or both. (g) FLAG IP in lysates from overexpression of Cas1-FLAG and Cas2 E9A-HA. (h) DNA affinity precipitations in the same lysates as in (g) using the same DNA constructs as in (e).

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