Fitting CRISPR-associated Cas3 into the helicase family tree

Abstract

Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies.

Figure 1. Structures and unwinding mechanisms of helicase superfamilies

(a) A schematic of the core helicase domain. The N-terminal RecA domain (RecA1) is represented by a blue cylinder and the C-terminal RecA domain (RecA2) is shown as a red cylinder. Conserved amino acid motifs are colored according to helicase function. Motifs in yellow are involved in NTP binding/hydrolysis, green are associated with translocation, and blue interact with nucleic acid. Motifs that are unique to specific superfamilies are highlighted with a red oval. The Walker A (A), Walker B (B) and arginine finger (R) motifs are conserved across all helicase superfamilies. (b) Topology diagrams depicting the secondary structure of the tandem RecA-like folds observed in SF1 and SF2 helicases. The RecA-like domains form a cleft that contains an NTP binding pocket (yellow) and a nucleic acid binding site (blue). NTP binding and hydrolysis causes the cleft to cycle between the closed and open states. (c) SF3-6 helicases assemble into toroidal hexamers that radially array the bipartite NTP binding sites. (d) Schematic of the unwinding mechanism for SF1 and SF2 helicases. The top and bottom panels represent closed (NTP-bound) and open (unbound) states, respectively. The RecA-like domains and conserved motifs are colored as in (a). NTP-dependent conformational changes drive a wedge (colored pink) between the oncoming strands of a duplex. (e) A schematic of the unwinding mechanism of the flat hexameric SF3 and SF5 helicases. The flat ring is depicted as a rectangle. The translocation strand threads through a central pore in the hexamer. The top panel shows the nucleic acid binding loops arranged in a spiral staircase configuration. The bottom panel depicts a downward motion of the top loop (blue wedge), during NTP binding and hydrolysis. (f) Schematic of the SF4 and SF6 hexamer bound to ssDNA and nucleotides before (top panel) and after (bottom panel) a NDP release. NTP binding at an empty site coupled with ADP release at an adjacent site moves the top domain of the lock washer in a 5’ to 3’ direction.

Figure 2. Fitting Cas3 into the helicase family tree

(a) Sequence logos of the conserved motifs in the core helicase domain of SF1, SF2 and Cas3 proteins. Green and blue boxes denote motifs that define Cas3 as SF2 helicases. Cas3 contains a unique motif (IV, red circle) not observed in other SF2 helicases. (b) Phylogenetic tree including 265 sequences representing SF1-6 helicases. Amino acid sequences from the core RecA helicase domains of 68 different Cas3 proteins were aligned to the helicase core domains of representative sequences from all superfamilies. Sequences were aligned with Clustal Omega and manually curated in Se-Al (see Supplementary Figure 1 for alignment). N- and C-terminal accessory domain sequences were not included. The alignment contained 878 amino acid positions, 572 of which are parsimony-informative (i.e. the position had at least one alternative amino acid in more than one sequence). Phylogenetic analysis was carried out with a Bayesian approach using MrBayes [54]. Tree topologies were sampled every 250 generations for 10⁶ generations using the WAG evolutionary model with fixed amino acid frequencies and gamma-shape rate variation with a proportion of invariable sites as recommended by ProtTest [55]. Posterior probabilities for all of the marked clades ranged 0.95-1.00.

Figure 3. Cas3 proteins form well-supported clades that support previously delineated CRISPR subtypes

(a) Schematic of the helicase core and accessory domains commonly observed in each Cas3 subtype. Conserved helicase motifs are colored as in Figure 1. Cas3 sequences in the Q motif and motif II (i.e. Walker B) can be used to delineate Cas3 subtype association. (b) Phylogenetic tree of the core helicase domains from 68 different Cas3 proteins. Alignments and phylogenies were performed as described in Figure 2. Bacterial sequences are colored red, archaeal sequences blue and viral sequences black. Posterior probabilities for all of the marked clades ranged 0.95-1.00.

Figure 4. The central role of Cas3 in CRISPR-associated adaptive immunity

CRISPR-mediated immunity proceeds in three basic steps: acquisition, crRNA biogenesis, and interference. Fragments of foreign DNA (protospacers) are acquired from regions of the invading genome that are flanked by short sequence motifs called protospacer adjacent motifs (PAMs). Protospacers are inserted into the CRISPR locus between direct repeats (black squares) by a mechanism that involves Cas1 and Cas2 proteins. The CRISPR locus is transcribed (pre-crRNA) and processed (little red arrows) into small crRNAs that are loaded into a crRNA-guided surveillance complex called Cascade (blue oval). Cascade is anticipated to facilitate target detection by scanning, and target recognition results in R-loop formation. The target bound surveillance complex recruits the effector nuclease-helicase Cas3 through a mechanism that is enhanced by ATP. Cas3 binds the R-loop and nicks the displaced strand. In the presence of ATP, Cas3 unidirectionally degrades the DNA target in a 3’ to 5’ direction. Cas3-mediated degradation may serve as a signal that recruits Cas1 and Cas2, resulting in the rapid acquisition of new spacers derived from the target strand of the DNA invader. This phenomenon is called priming.