Structural insights of DNA slicing and dicing by PaqCI restriction enzyme

From the Stoddard Lab, Basic Sciences Division

Enzymes that cut DNA at specific sites are called endonucleases; these enzymes play many roles in genomic replication, fidelity, and defense. The initial discovery of restriction endonucleases in the 1950s significantly enhanced our understanding of such enzymes and enabled recombinant DNA technologies, cloning, and gene editing. Interestingly, the role of restriction endonucleases was first discovered as a defense mechanism of bacteria against foreign viral DNA called phage. The “Type IIS” restriction endonucleases, a branch of such bacterial enzymes, typically have two separate protein domains: one to recognize a specific nucleotide sequence and another to cleave or cut the DNA at a well-defined distance from the recognition site. This domain separation enables retention of the 4-7 nucleotide recognition site following DNA cleavage, a useful feature in DNA synthesis techniques such as Golden Gate assembly that in some cases can assemble more than 50 DNA fragments at once in an ordered fashion. To continue characterizing these Type IIS restriction enzymes and to hone their uses for biotechnology, Madison Kennedy, a graduate student in Dr. Barry Stoddard’s lab, sought to refine to atomic resolution the structure of DNA-bound PaqCI Type IIS restriction enzyme complexes during DNA cleavage. Their work, which was done in collaboration with investigators at New England Biolabs, was published recently in Nucleic Acids Research.

The orientation and mechanism of how these types of restriction endonucleases operate remains unclear. To provide insight into their operation, Drs. Kennedy and Stoddard had two aims in this study: “The first is the manner in which many of them [restriction enzymes] only become licensed to cleave and degrade DNA when they pull multiple, unprotected target sites together into a reaction synapse [as depicted in the diagram below]. That process is critical to their ability to distinguish between being engaged to the host’s DNA (which does not get degraded) versus being bound to foreign DNA (which contains multiple unprotected sites) which is then destroyed by the enzyme. We and others have been trying for many years (about 10 in our case) to visualize this process, but only succeeded recently when [cryoelectron microscopy] CryoEM became available to us. Prior to that, attempts to isolate and crystallize such complexes, which are highly dynamic, had consistently failed.”

“The second is the manner in which Type IIS enzymes cleave DNA,” explained Dr. Stoddard. “This has been of great interest to many investigators involved in targeted gene disruption and modification, because two important types of gene targeting systems (zinc finger nucleases or ‘ZFNs' and TAL effector nucleases or 'TALENs') incorporate the catalytic domains of such enzymes into their architectures.” In other words, these gene-editing nucleases are engineered for enhanced specificity, and with an increased understanding of how these enzymes function, the field may continue to hone editing specificity in both in vitro and in vivo systems.

(Left) Diagram of two PaqCI monomers recognizing specific DNA sequences and the formation of DNA loops to enable DNA cleavage. (Right) CryoEM structure of a PaqCI tetramer bound to DNA (grey) at 4+ sites for DNA recognition and cleavage reaction.
(Left) Diagram of two PaqCI monomers recognizing specific DNA sequences and the formation of DNA loops to enable DNA cleavage. (Right) CryoEM structure of a PaqCI tetramer bound to DNA (grey) at 4+ sites for DNA recognition and cleavage reaction. Images were provided by Drs. Kennedy and Stoddard

To provide insight into these areas of ambiguity, the Stoddard lab employed cutting-edge cryological electron microscopy (‘CryoEM’) to visualize the structure of PaqCI-DNA complexes at the stage of DNA cleavage. This process required purification of the endonuclease and optimization of PaqCI-mediated DNA cleavage conditions for the lambda phage template DNA, an arduous process even for skilled researchers. Next, CryoEM structures of PaqCI complexes were resolved in the absence and presence of phage DNA. Under both conditions, the PaqCI enzyme displayed a tetrameric structure. The researchers also observed major structural differences in the two PaqCI monomers (red and green protruding structures in the figure on the lower region of the tetramer) that engaged the phage DNA to mediate cleavage. These CryoEM structures provided new insights into the structural orientation of the initial PaqCI complex and changes that occur following enzyme interactions with foreign DNA.

“This study (and another that we recently published, on the structure of a different restriction/modification enzyme called “DrdV”) demonstrates that enzymes that generate cleavage assemblages from multiple bound DNA target sites can do so with very different structural mechanisms, that would lead to significantly different target search mechanisms and kinetics,” summarized Dr. Stoddard. As mentioned above, the role of restriction endonucleases is to protect bacteria against foreign DNA, including phage DNA. “Whether such differences [in mechanisms of cleavage assemblages] correspond to unique biological properties or capabilities of such enzymes, that would make them especially efficient at defending against certain types of phage is unknown,” concluded Dr. Stoddard. But this is an area they would like to explore. Specifically, Dr. Stoddard stated, “We hope that our next stage of research will examine the mechanism by which these types of enzymes can broaden their specificities (and therefore target additional phage DNA sequences) by a mechanism called ‘phase variation’, in which alternative translated gene products from the same gene correspond to enzymes with altered specificities.”

Lastly, Dr. Stoddard highlighted key Fred Hutch funding for this research: “The Cancer Consortium has enabled this research both through direct support of our lab via discretionary funding for pilot projects such as this one” and “through significant support of the Center’s new Cryoelectron Microscopy facility.”

The spotlighted research was funded by the National Institutes of Health, Fred Hutchinson Cancer Center, New England Biolabs, US National Science Foundation, and the U.S. Department of Energy.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Barry Stoddard contributed to this work.

Kennedy MA, Hosford CJ, Azumaya CM, Luyten YA, Chen M, Morgan RD, Stoddard BL. 2023. Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI. Nucleic Acids Res. 51(9):4467-4487.