An EM View of the FokI Synaptic Complex by Single Particle Analysis

Abstract

FokI is a Type IIS restriction endonuclease that recognizes the 5′-GGATG-3′ sequence and cleaves non-specifically at 9 and 13 base pairs away on the top and bottom strands respectively to produce a 5′ overhang. FokI is a bipartite endonuclease with separate recognition and cleavage domains. Because of its bipartite nature, FokI has received considerable interest in generating chimeric nucleases for use in biotechnology, and recently as possible therapeutic agents in gene therapy by initiating homologous gene recombination and repair. Here we show, using single-particle electron microscopic studies, that the FokI active complex prefers a single conformation in which the subunits are arranged in a doughnut shape complex with protein-protein and possibly protein-DNA interactions stabilizing the cleavage complex. Our EM model provides new insights into the activation mechanism of FokI and how non-specific cleavage is avoided. Keywordsbipartite restriction endonuclease; DNA recognition and cleavage; synaptic complex; single particle analysis; electron microscopy The Type IIS restriction endonuclease FokI cleaves DNA non-specifically at a fixed distance of 9 and 13 nucleotides downstream of a non-palindromic recognition sequence 5′-GGATG-3′. The enzyme is monomeric (~66 kDa) in solution and has a modular structure consisting of an N-terminal DNA recognition domain and a C-terminal cleavage domain 1 . The bipartite nature of FokI has made it an excellent candidate for the design of hybrid endonucleases with novel sequence specificities, by attaching the cleavage domain of FokI to the DNA binding domains of transcription factors such as zinc fingers and homeodomains 2-4 . In particular, the chimeric zinc finger nucleases (ZFNs) have shown potential as therapeutic agents in gene targeting by initiating homologous gene recombination and repair of damaged DNA 4-9 .Despite the considerable interest in generating novel nucleases and therapeutics using FokI, a picture of the mechanism of cleavage of the native enzyme has not been fully achieved. An important step toward this goal was the determination of the crystal structure of enzyme bound to its cognate DNA 10 . The structure determined with a 20-mer oligonucleotide in the absence * Corresponding author. E-mail address: E-mail: eva.vanamee@mssm.edu. # Present address: The National Institute for Medical Research, The Ridgeway. Mill Hill, London, NW7 1AA, UK Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. of divalent metals, showed that the cleavage domain was packed alongside the recognition domain and hence was positioned aw...

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

“…In the other FokI structure, without DNA, the catalytic domains of two subunits—both in sequestered conformation—pair to form a catalytic center for dsDNA cleavage. Modeling and electron microscopy ( 41 , 45 ) suggest that at the time of DNA cleavage, one of the FokI monomers adopts an alternative conformation that brings its catalytic domain close to the DNA molecule to which it is bound. Following transient dimerization, this domain then hydrolyzes the bottom DNA strand 13 bases from the recognition sequence, and the other, ‘recruited’ domain, which remains sequestered, hydrolyzes the top strand 9 bases away ( 46 ).…”

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

“…Complexes were visualized in a carbon-sandwich uranyl formate negative-stain preparation, as described by Ohi et al (20). Even though DNA has weaker contrast than protein in negative stain (21)(22)(23)(24), this preparation method has yielded the most structural detail thus far of the complex. Threedimensional (3D) reconstructions from negative stain typically yield accurate (to within a few Å) localization of protein domain centers relative to each other and permit mapping of molecular interaction sites at a 10-20 Å resolution level (25).…”

“…2 B and D]. Weaker protein density is interpreted to arise from positional and/or occupancy variability of the affected regions within the particle population; weaker DNA density is expected, owing to reduced contrast (21)(22)(23)(24).…”

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

“…Cryo-EM studies of FokI bound to short oligoduplexes revealed a croissant-shaped object that could be reconciled more readily to the parallel rather than the anti-parallel alignment (45). However, the synapsis of sites in cis may be governed by other factors.…”