Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

Figure 1

(A) Schematic organization of the CRISPR4/Cas system of S. thermophilus DGCC7710 and the CRISPR/Cas system of E. coli K-12. Percentage of identical and similar (in parenthesis) residues between corresponding protein sequences that are connected by dashed lines. Conserved repeat sequences of each system are shown in the inserts. (B) Domain architecture of the S. thermophilus Cas3 protein. Domains identified by in silico analysis are shown as grey boxes. HD domain denotes HD-type phosphohydrolase/nuclease domain; DExD/H domain denotes DExD/H-box helicase domain; HelC dom denotes the C-terminal helicase domain. Conserved residues characteristic of the different domains and subject to alanine mutagenesis are indicated above the boxes. Location of the conserved helicase motifs are indicated by numbers I, II and VI.

Figure 2

Cas3 ATPase activity. (A) Radioactive ATPase assay. ATPase reactions were conducted at 30°C in a reaction buffer containing 10 mM Tris–HCl (pH 7.5 at 25°C), 30 mM KCl, 5% (v/v) glycerol, 2 mM MgCl₂, 0.1 mg/ml BSA, 0.5 mM ATP, 2.5 nM ssDNA (M13mp18) or dsDNA (supercoiled form of pUC57 plasmid), and 250 nM Cas3. Reaction mixtures were supplemented with [α³²P]ATP (5 Ci/mmol), spotted onto a polyethyleneimine-cellulose thin-layer plate and separated by chromatography followed by phosphorimager visualization. (B) ATP hydrolysis dependence of nucleic acids. Malachite green assay was used to measure ATP hydrolysis through the detection of liberated-free phosphate from ATP. Reaction mixtures in the buffer described above contained varying amounts of ssDNA (M13mp18), dsDNA (supercoiled form of pUC57 plasmid) or 2223 nt RNA. (C) Time courses of ATP hydrolysis. Reaction mixtures contained 3 nM ssDNA (M13mp18). Malachite green assay was used to measure ATP hydrolysis through the detection of liberated-free phosphate from ATP. (D) ATP hydrolysis rates. Reaction rate constant k (min⁻¹) calculated from slopes of times courses shown in (C).

Figure 3

Cas3 nuclease activity. (A) Degradation of the ssDNA and dsDNA. Various amounts of Cas3 were incubated in the presence of 4 nM of M13 ssDNA or pUC57 dsDNA at 37°C for 2 h in the presence (+) or absence (−) of 10 mM MgCl₂ or 10 mM EDTA. (B) Effect of mutations on Cas3 nuclease activity. In all, 500 nM of protein was incubated in the presence of 4 nM of M13 ssDNA at 37°C for 2 h.

Figure 4

Cas3 helicase activity and polarity. (A). Schematic representation of duplex unwinding assay. (B, C) DNA–DNA and RNA–DNA duplex unwinding by Cas3. Cas3 displacement of a P³²-labelled 20 nt oligodeoxynucleotide (B) or 22 oligoribonucleotide (C) annealed to an ssM13mp18 DNA is monitored in the polyacrylamide gel. Reactions were performed at 30°C for 1 h in the reaction buffer: 10 mM Tris–HCl (pH 7.5 at 25°C), 25 mM KCl, 15% (v/v) glycerol, 1 mM MgCl₂, 0.1 mg/ml BSA, 2 mM ATP, 0.5 nM substrate and various amounts of protein. nA denotes the ATP analogue AMP-PNP. D452A and D77A are ATPase and nuclease domain mutants, respectively. (D) Cas3 polarity assay I. Cas3 displacement of 30 nt double-stranded fragments at the ends of the linear M13mp18 DNA. Partial duplex DNA was prepared by EheI cleavage of the labelled 60 nt oligodeoxynucleotide annealed to the M13pm18 DNA. Duplex regions are separated by a single-stranded region of few thousand nucleotides. Reaction mixture contained 500 nM of Cas3. Reactions were stopped at defined time intervals. (E) Cas3 polarity assay II. Cas3 displacement of the oligonucleotide-based 73 nt substrates containing 53 nt 3′- or 5′-overhangs. Reactions were performed as in Figure 4B and C except that with the oligonucleotide-based substrates 500 nM of Cas3, 1.5 nM of substrate and 250 nM trap DNA (unlabelled oligonucleotide) were used in the reaction.

Figure 5

Proposed Cas3 mechanism of action. Assembly of the ternary Cascade–crRNA–Cas3 complex with the target DNA promotes strand separation and crRNA hybridization to the complementary DNA strand to generate the R-loop structure. A single-DNA strand in the R-loop structure is cut at multiple sites by Cas3 nuclease resulting in the single-strand break in the protospacer region. After cleavage of one DNA strand, Cas3 helicase activity may further remodel the Cascade–crRNA complex, for example, by displacing the Cascade–crRNA, and create a platform for the second DNA strand cleavage by the HD domain to generate a double-strand break.