Abstract
BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5′-CCTCAGC-3′, generating 3-base, 5′-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).
Highlights
Restriction endonucleases (REases) function as an innate form of microbial immune system, distinct from the adaptive form exemplified by CRISPR–Cas nucleases [1]
Bp target sequences cleaved by CRISPR–Cas nucleases are ‘learned’ from prior exposure and largely recognized via RNA–DNA base pairing [2], whereas the target sequences cleaved by REases are much shorter, are recognized through protein–DNA interactions, and are intrinsic to the structure of each enzyme [3,4,5]
Earlier mutagenesis studies of BbvCI demonstrated that E167-V168-K169 in the R1 subunit, and E177-C178-K179 in the R2 subunit, comprise the distal residues of this motif (Figure 1A)
Summary
Restriction endonucleases (REases) function as an innate form of microbial immune system, distinct from the adaptive form exemplified by CRISPR–Cas nucleases [1]. Both act to disrupt invasive DNA molecules such as viral genomes that infect bacterial and archaeal cells. Restriction endonucleases span a wide range of protein folds, catalytic motifs, domain architectures and mechanisms [12,13]. This diversity is reflected in a system of classification that divides these enzymes into overlapping groups based upon subunit organization, target characteristics and cleavage positions [14]. Type II restriction enzymes and their methyltransferase partners are encoded by separate genes, and act independently of one another [20]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
