An EM view of the FokI synaptic complex by single particle analysis

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 electron microscopy (EM) model provides new insights into the activation mechanism of FokI and how non-specific cleavage is avoided.     

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.     

Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs

Type IIS restriction endonuclease BtsCI (GGATG 2/0) is a neoschizomer of FokI (GGATG 9/13) and cleaves closer to the recognition sequence. Although M.BtsCI shows 62% amino acid sequence identity to M.FokI, BtsCI and FokI restriction endonucleases do not share significant amino acid sequence similarity. BtsCI belongs to a group of Type IIS restriction endonucleases, BsmI, Mva1269I and BsrI, that carry two different catalytic sites in a single polypeptide. By inactivating one of the catalytic sites through mutagenesis, we have generated nicking variants of BtsCI that specifically nick the bottom-strand or the top-strand of the target site. By treating target DNA sequentially with the appropriate combinations of FokI and BtsCI nicking variants, we are able to generate long overhangs suitable for fluorescent labeling through end-filling or other techniques based on annealing of complementary DNA sequences.     

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer

The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step.     

Overproduction and crystallization of FokI restriction endonuclease.

To overproduce FokI endonuclease (R.FokI) in an Escherichia coli system, the coding region of R.FokI predicted from the nucleotide sequence was generated from the FokI operon and joined to the tac promoter of an expression vector, pKK223-3. By introduction of the plasmid into E. coli UT481 cells expressing the FokI methylase gene, the R.FokI activity was overproduced about 30-fold, from which R.FokI was purified in amounts sufficient for crystallization. The removal of a stem-loop structure immediately upstream of the R.FokI coding region was essential for overproduction.     

Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting ‘top’ and ‘bottom’ strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

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

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology.     

M.FokI methylates adenine in both strands of its asymmetric recognition sequence

M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.     

Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties

Class IIS restriction endonucleases cleave double-stranded (ds) DNA at precise distances from their recognition sequences. A method is proposed which utilizes this separation between the recognition site and the cut site to allow a class IIS enzyme, e.g., FokI, to cleave practically any predetermined sequence by combining the enzyme with a properly designed oligodeoxynucleotide adapter. Such an adapter is constructed from the constant recognition site domain (a hairpin containing the ds sequence, e.g., GGATG CCTAC for FokI) and a variable, single-stranded (ss) domain complementary to the ss sequence to be cleaved (at 9 and 13 nucleotides on the paired strands from the recognition sequence in the example of FokI). The ss sequence designated to be cleaved could be provided by ss phage DNA (e.g., M13), gapped ds plasmids, or supercoiled ds plasmids that were alkali denatured and rapidly neutralized. Combination of all three components, namely the class IIS enzyme, the ss DNA target sequence, and the complementing adapter, would result in target DNA cleavage at the specific predetermined site. The target ss DNA could be converted to the precisely cleaved ds DNA by DNA polymerase, utilizing the adapter oligodeoxynucleotide as primer. This novel procedure represents the first example of changing enzyme specificity by synthetic design. A practically unlimited assortment of new restriction specificities could be produced. The method should have many specific and general applications when its numerous ramifications are exploited.     

The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.     

Sequence analysis of Vicia faba repeated DNA, the FokI repeat element

A type of highly repeated DNA sequences present in the genome of Vicia faba was detected by digestion its nuclear DNA with FokI endonuclease and fractionating the digests on polyacrylamide gels. Four fragments of 59, 108, 177 and 246 bp of the FokI repeated sequences were collected from the gels and their primary structures were determined by the method of Maxam and Gilbert. These repeated DNA sequences were shown to be a multiple tandem array of a 59 bp sequence element. And its nucleotide sequence was almost completely conserved among all the sequence members of each the size class and also among these classes. This sequence element consists of a duplet of an about the duplet has an incomplete dyad symmetrical structure.     

FokI method of gene synthesis

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.     

Specificity of DNA binding and methylation by the M.FokI DNA methyltransferase

The M.FokI adenine-N(6) DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine-N(6) DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2-3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein-protein interaction assays show that both domains of M.FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M.FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.     

Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.     

利用mtCOI PCR-RFLP技术鉴定中国境内九个烟粉虱隐种

烟粉虱Bemisia tabaci (Gennadius)是一个物种复合体, 包括31个以上形态上无法区分的隐种, 其中少数隐种是世界性入侵害虫。目前, 在中国境内分布有2个入侵隐种和13个土著隐种。快速、 高效的鉴别方法对掌握烟粉虱田间发生规律及制定相关防控策略具有重要意义。然而到目前为止, 除了mtCOI基因测序比对外, 尚未有一种简便的方法可以有效地区分多个烟粉虱隐种。本研究采用mtCOI PCR-RFLP技术, 单独或组合使用TaqI, VspI, Van91I, NcoI和FokI这5种限制性内切酶, 酶切烟粉虱mtCOI的PCR扩增片段, 鉴别分布在中国境内的9个烟粉虱隐种。结果表明, 单独使用TaqI酶切, 可鉴别出MEAM1和China 1两个隐种, 使用TaqI+NcoI分步酶切可鉴定出MED和Asia 1两个隐种, 使用TaqI+Van91I分步酶切可鉴定出Asia II 3和Asia II 9两个隐种, 使用TaqI+VspI+FokI分步酶切可鉴定出Asia II 1, Asia II 6和Asia II 7 3个隐种。本研究为高效鉴别中国境内的多个烟粉虱隐种提供了方法。