Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family.

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4-8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5'-CCNGG flip the central N nucleotides and 'compress' the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5'-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not 'compress' the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences

The H-subunit of the restriction endonuclease CglI contains a prototype DEAD-Z1 helicase-like motor

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG (“/” designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences

Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits

Summary: The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function. We show that the Cas10-Csm4 subcomplex binds the 5′-handle of crRNA and triggers Csm3 oligomerization to form a padlock for crRNA binding. We demonstrate that Csm5 plays a key role in target RNA binding while Csm2 ensures RNA cleavage at multiple sites by Csm3. Finally, guided by deletion analysis, we engineered a minimal Csm complex containing only the Csm3, Csm4, and Cas10 subunits and crRNA and demonstrated that it retains all three catalytic activities, thus paving the way for practical applications. : Mogila et al. characterized individual subunits of the CRISPR-Cas type III-A Csm effector complex to determine their role in complex assembly and function. A designed minimal Csm complex composed of three subunits (Cas10, Csm3, and Csm4) and a crRNA retains all the functions of the wild-type Csm complex. Keywords: CRISPR-Cas, Csm effector complex, Cas10, Csm2, Csm3, Csm4, Csm5, Cas6, minimal Csm comple

Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5′-W/CCGGW-3′ (W stands for A or T, ‘/’ denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an ‘open’ configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG (“/” designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences