Insertion and deletion mutants of FokI restriction endonuclease

The present invention reveals the construction of several insertion (4, 8, 12, 18, 19 or 23 amino acid residues) and deletion (4 or 7 amino acid residues) mutants of the linker region of FokI endonuclease in Flavobacterium okeanokoites. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence-specificity as the wild-type enzyme. However, compared with the wild-type enzyme, the insertion mutants cleave predominantly one nucleotide further away from the recognition site on both strands of the DNA substrate. The four codon deletion mutant shows relaxed specificity at the cut site while the seven codon deletion appears to inactivate the enzyme. The DNA-binding and cleavage domains of FokI appear to be linked by a relatively malleable linker. No simple linear relationship exists between the linker length and the distance of the cut site from the recognition site. Furthermore, the four codon insertion mutants cleave DNA substrates containing hemi-methylated FokI sites; they do not cleave fully-methylated substrates.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the construction of six insertion (4, 8, 
12, 18, 19 or 23 amino acid residues) and two deletion (4 or 7 amino acid 
residues) mutants of the linker region of FokI endonuclease from 
Flavobacterium okeanokoites. The mutant enzymes were purified, and their 
cleavage properties have been characterized. 
2. Description of the Related Art 
The FokI restriction endonuclease from Flavobacterium okeanokoites belongs 
to the Type IIS class of endonucleases. FokI recognizes the asymmetric 
sequence 5'-GGATG-3' and cleaves double-stranded DNA at staggered sites 9 
and 13 nucleotides away from the recognition site (1, 2). The cloning and 
sequencing of the FokI restriction-modification system have been reported 
(3-4). Several research groups have purified FokI endonuclease and 
characterized its properties (5-9). Previous reports by the present 
inventor on proteolytic fragments of FokI endonuclease using trypsin have 
revealed a N-terminal DNA-binding domain and a C-terminal catalytic domain 
with non-specific DNA cleavage activity (10,11). These reports have 
suggested that the two domains are connected by a linker region which is 
susceptible to cleavage by trypsin. The present inventor has also shown 
that insertion of four (or seven codons) between the recognition and 
cleavage domains of FokI can alter the cleavage distance of FokI within 
its substrate (12). 
Recently, Waugh and Sauer have shown that single amino acid substitutions 
uncouple the DNA-binding and strand scission activities of FokI 
endonuclease (13). Furthermore, they have obtained a novel class of FokI 
restriction mutants that cleave hemi-methylated DNA substrates (14). The 
modular structure of FokI suggested that it may be feasible to construct 
hybrid endonucleases with novel sequence-specificity by linking other 
DNA-binding proteins to the cleavage domain of FokI endonuclease. 
Recently, the present inventor reported the construction of the first 
"chimeric" restriction endonuclease by linking the Ubx homeo domain to the 
cleavage domain of FokI (15). 
To further probe the linker region, the present inventor has constructed 
several insertion and deletion mutants of FokI endonuclease. A detailed 
description of the process for making and using and the properties of 
these mutants follows. 
SUMMARY OF THE INVENTION 
The present invention discloses the construction of seven insertion (4, 4, 
8, 12, 18, 19 or 23 amino acid residues) and two deletion (4 or 7 amino 
acid residues) mutants of the linker region of FokI endonuclease from 
Flavobacterium okeanokoites. The FokI endonuclease has an N-terminal DNA 
recognition domain, a C-terminal DNA cleavage domain and a linker region 
between these domains. 
The insertion mutant FokI endonucleases of the present invention are 
characterized in that the distance recognized between the recognition site 
and the cleavage site of the DNA substrate is shifted one base pair away 
from the recognition site as compared to the distance between the 
recognition site and the cleavage site of the DNA substrate when wild-type 
enzyme is used. 
The insertion mutants of the present invention are also characterized by 
having specific amino acid residues inserted between the recognition 
domain and the cleavage domain of the endonuclease. 
The deletion mutants of the present invention are characterized by having 
specific amino acid residues deleted between the recognition domain and 
the cleavage domain of the endonuclease.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention details the construction of seven insertion mutants 
(4, 4, 8, 12, 18, 19 or 23 amino acid residues) and two deletion mutants 
(4 or 7 amino acid residues) of the linker region of FokI endonuclease 
from Flavobacterium okeanokoites. 
The insertion mutants have an amino acid residue insertion between the 
recognition domain and the cleavage domain where the insertion is selected 
from the group consisting of SEQ ID NO:3, 4, 5, 6, 7, 8 and 9. 
The deletion mutants have an amino acid residue deletion between the 
recognition domain and the cleavage domain where the deletion consists of 
KSEL (SEQ ID NO:3) or KSELEEK (SEQ IN NO:14). 
The insertion and deletion mutant endonucleases of the present invention 
were prepared and characterized as follows: 
EXPERIMENTAL PROCEDURES 
E. coli RR1 strain was the host in all experiments. RR1[pACYCfokIM] which 
carries a single copy of the FokI methyltransferase gene (fokIM) was used 
for the construction and expression of the insertion and deletion mutants 
of FokI endonuclease. 
The mutant genes were cloned into pRRS under the control of lacUV5 
promoter. The detailed structure of pRRSfokIR and pACYCfokIM have been 
published previously (10). The pTZ19R plasmid DNA was used as the 
substrate to assay the activity of the mutant enzymes. 
Construction of insertion and deletion mutants of FokI endonuclease 
The PCR technique was used to insert or delete amino acid residues within 
the proposed linker region of FokI endonuclease as previously reported 
(12). The PCR generated DNA containing the insertions or deletions were 
digested with SpeI/SmaI and gel-purified. The plasmid pRRSfokIR that 
contains a single SpeI site within the fokIR gene was cleaved with 
SpeI/SmaI, and the large 3.9-kb fragment was gel-purified and ligated to 
the PCR product. 
After selection on LB plates containing tetracylin (20 .mu.g/ml) and 
ampicillin (60 .mu.g/ml), the plasmid DNA from the surviving clones were 
screened for larger SpeI/HindIII inserts as compared to the wild-type 
fragment by agarose (1.5%) gel electrophoresis. The cultures of the 
positive clones were induced with isopropyl-.beta.-D-thiogalactopyranoside 
(IPTG). After sonication, the mutant enzymes were partially purified using 
a phosphocellulose column and then were assayed for restriction 
endonuclease activity. 
In addition, the recombinant plasmids from the active clones were isolated 
and the presence of the insertions or deletions were confirmed by Sanger's 
dideoxy sequencing method (16). The construction of the RGGGGSGGGGSGGGGSQL 
(SEQ ID NO:9) insertion mutant containing the glycine linker (Gly.sub.4 
Ser).sub.3 (SEQ ID NO:15) was done by synthesizing the appropriate 
complementary oligonucleotides. The oligonucleotides were annealed to 
yield the duplex shown in FIG. 1. The duplex is flanked by XbaI and SpeI 
restriction sites. The annealed duplex was filled-in by using dNTPs and 
Klenow fragment to generate the XbaI and SpeI sites. The duplex was then 
digested with SpeI/XbaI, purified using G-25 spun column and then ligated 
into SpeI-cleaved pRRSfokIR plasmid. 
The recombinants were screened for appropriate inserts using 
SpeI/ScaI/HindIII enzyme digestion. The presence of the insert in the 
right orientation within the gene was confirmed by using Sanger's dideoxy 
sequencing method (16). 
Overproduction and purification of the mutant enzymes 
The procedures for cell growth and purification of the mutant enzymes are 
similar to the one used for the wild-type FokI and the four (or seven) 
codon insertion mutants that have been previously reported (12). The 
purity of the proteins were analyzed by 0.1% SDS-12% acrylamide gel 
electrophoresis (17), followed by staining the protein with coomassie blue 
(FIG. 2). 
Preparation of DNA substrates with a single FokI site 
Substrate containing a single FokI site was constructed by forming a duplex 
using synthetic oligonucleotides SEQ ID NO:10 and SEQ ID NO:11 as shown in 
FIG. 3. The oligomers were phosphorylated and then annealed to form the 
duplex. The duplex is flanked by XbaI and HindIII compatible ends. The 
fragment was ligated to XbaI/HindIII-cleaved pTZ19R plasmid. The 
recombinants were screened for the insert using XbaI/HindIII digestion. 
A 300-bp fragment containing the insert was gel-purified after digesting 
the recombinant plasmid with PuvII enzyme. The fragment was 
dephosphorylated by treatment with calf intestinal phosphatase (CIP) and 
then was rephosphorylated using T4 polynucleotide kinase and 
[.beta..sup.32 -P] ATP to obtain substrate that was labelled on both 
strands of the DNA. Digestion of the above labelled fragment with either 
HinPI or XbaI, followed by gel-purification of the appropriate DNA 
fragment, yielded substrates that were labelled individually on each 
strand. 
The cleavage assays with plasmid substrates were performed by adding pTZ19R 
(1.5.mu.g) and the enzyme (40 nM) in a total reaction volume of 15 .mu.l 
containing 10 mM Tris.HCl (pH 7.5 at 37.degree. C.), 50 mM NaCl and 10 mM 
MgCl.sub.2 and 1 mM DTE. The reactions were incubated at 37.degree. C. for 
4 hrs. The digests were analyzed by agarose gel (1.2%) electrophoresis 
(FIG. 4). For determining the cleavage distance from the recognition site 
within the DNA substrate, .sup.32 P-labelled substrates (.congruent.10,000 
cpm) were mixed with 0.5 .mu.g of pTZ19R and 45 nM of enzymes in a total 
volume of 10 .mu.l containing 10 mM Tris. HCl (pH 7.5 at 37.degree. C.), 
50 mM NaCl, 10 mM MgCl.sub.2 and 1 mM DTE. The reaction mixture was 
incubated at 37.degree. C. for 2 hrs. The samples were subjected to 
electrophoresis on 7% PAGE containing 7M urea in 1 x TBE, the gel dried, 
and then exposed to x-ray film (FIGS. 5 and 6). 
Cleavage of Hemi-Methylated DNA Substrates 
The four oligonucleotides (SEQ ID NO:12, SEQ ID NO:13, hemi-methylated 
oligonucleotide of SEQ ID NO:12 and hemi-methylated oligonucleotide of SEQ 
ID NO:13) used in these experiments are the same oligonucleotides that 
were used by Waugh and Sauer to show that two of their eight FokI 
endonuclease missense mutants cleave hemi-methylated DNA substrates (14). 
Each of the four oligomers were phosphorylated using T4 polynucleotide 
kinase and [.beta..sup.32 -P] ATP. By annealing various combinations of 
the synthetic substrates, it was possible to construct an unmethylated 
substrate, two hemi-methylated substrates and a fully methylated 
substrate. 0.02 picomole of each labelled substrate and about 70 nM sites 
(pTZ19R) were mixed with 70 nM of FokI or insertion mutants, KSEL (SEQ ID 
NO:3) or TAEL (SEQ ID NO:4), in 15 .mu.l of reaction buffer described 
above. The samples were analyzed on 9% PAGE containing 7 M urea, the gel 
was dried and then exposed to an x-ray film (FIG. 7). 
Filter Binding Assays 
The filter binding assays were performed in duplicate using FokI or mutant 
enzymes and the four different synthetic substrates, SEQ ID NO:12, SEQ ID 
NO:13, hemi-methylated oligonucleotide of SEQ ID NO:12 and hemi-methylated 
oligonucleotide of SEQ ID NO:13. Nine different concentrations for each of 
the enzymes were made by serial dilutions (5000, 2000, 667, 222, 74.1, 
24.7, 8.23, 2.74 and 0.914 nM). The enzymes were mixed with 40 nM of each 
labelled substrate along with 50 .mu.g/ml p(dI-dC) and 50 .mu.g/ml BSA in 
50 .mu.l of buffer A[10 mM Tris. phosphate (pH 8.0), 7 mM 
2-mercaptoethanol, 1 mM EDTA, 50 mM NaCl, and 10% (vol/vol) glycerol]. The 
samples were incubated at 22.degree. C. for 1 hr and then loaded on to 
nitrocellulose filters. The filters were washed twice with 0.5 ml of 
buffer A, dried and counted using the scintillation counter. 
Construction of Insertion and Deletion Mutants of FokI Endonuclease 
Previously, the present inventor has shown that introduction of additional 
amino acid residues (four or seven residues) between the recognition and 
cleavage domains of FokI can alter the spacing between the recognition 
site and the cleavage site within the DNA substrate (12). Secondary 
structure prediction of FokI endonuclease based on its primary amino acid 
sequence revealed a long stretch of .alpha.-helix region at the junction 
of the recognition and cleavage domains. If the helix constituted the 
linker that connects the two domains of the enzyme, the cleavage distance 
of FokI from the recognition site could be altered by changing the length 
of this spacer. Insertion of either four codons or seven codons into the 
linker region of FokI was expected to shift the cleavage distance one bp 
and two bp respectively away from the recognition site. Close examination 
of the amino acid sequence of the linker region revealed the presence of 
two KSEL (SEQ ID NO:3) repeats separated by amino acids EEK. Therefore, 
KSEL (SEQ ID NO:3) and KSELEEK (SEQ ID NO:14) were inserted within the 
linker region of FokI. Both mutants cleaved DNA in a similar way and not 
necessarily in a distance-dependent way (12). Thus, a clear relationship 
between the length of the connector region of FokI and the cleavage 
distance from the recognition site within its DNA substrate could not be 
established. 
A direct relationship has been shown between the length of the protein 
connector regions of EcoR124 and EcoR124/3 (belonging to the Type I class) 
and their related but different recognition site 5'-GAA(N.sub.6)RTGG--3' 
(SEQ ID NO:16) and 5'-GAA(N.sub.7)RTGG-3' (SEQ ID NO:17), respectively, 
where R=G or A and N=G, A, T, or C. The recognition sites differ only in 
the length of the non-specific spacer. This difference nevertheless places 
the two specific domains of the EcoR124/3 sequence 3.4 .ANG. further apart 
and rotates them 36.degree. with respect to those of EcoR124, which 
implies major structural differences in the proteins recognizing the 
sequences (18). This is accommodated in the protein structure by altering 
the number of amino acid repeats (TAEL).sub.2 (SEQ ID NO:5) and 
(TAEL).sub.3 (SEQ ID NO:6), respectively, within the connector region 
(18). 
To further probe the structure of the linker region between the recognition 
and cleavage domains of FokI endonuclease, a series of mutants with 
various number of amino acid residue deletions or insertions ranging from 
-7 to +23 residues was constructed (Table I). The amino acid segments KSEL 
(SEQ ID NO:3), TAEL (SEQ ID NO:4) and KSELEEK (SEQ ID NO:14) were used as 
basic units of insertion or deletion. As indicated above, the TAEL (SEQ ID 
NO:4) segment was observed in the protein connector regions of EcoR124 and 
EcoR124/3 (Type I) enzymes. Multiples of the basic units and a combination 
thereof were inserted between the recognition and catalytic domains of 
FokI to form the mutants. The method used to construct the insertion and 
deletion mutants are the same as the one previously reported (12). In 
addition, an 18 amino acid residue insertion mutant that includes the 
glycine linker, (Gly.sub.4 Ser).sub.3 (SEQ ID NO:15) was constructed. The 
clones of each mutant were induced with 1 mM IPTG for 4-5 hrs for optimal 
expression of the enzymes. The mutant enzymes were purified using the 
procedure previously described (10, 12). SDS/PAGE profiles of the mutant 
enzymes are shown in FIG. 2. 
TABLE I 
__________________________________________________________________________ 
Insertion/deletion mutants of FokI restriction endonuclease 
Number of 
aa sequence at the insertion/deletion site.sup.1 
aa insertion 
Activity.sup.2 
__________________________________________________________________________ 
. . . QLVKSELRHKLK . . . SEQ ID NO: 18 -7 
- 
. . . QLVEEKKSELRHKLK . . . SEQ ID NO: 19 -4 
+ 
. . . QLVKSELEEKKSELRHKLK . . . SEQ ID NO: 20 wt 
+ 
. . . QLV TAELKSELEEKKSELRHKLK . . . SEQ ID NO: 21 +4 
+ 
. . . QLVTAELTAELKSELEEKKSELRHKLK . . . 
SEQ ID NO: 22 +8 
+ 
. . . QLVTAELTAELTAELKSELEEKKSELRHKLK . . . 
SEQ ID NO: 23 +12 
+ 
. . . QLVTAELTAELKSELKSELEEKKSELEEKKSELRHKLK . . . 
SEQ ID NO: 24 +19 
+ 
. . . QLVTAELTAELTAELKSELKSELEEKKSELEEKKSELRHKLK . . . 
SEQ ID NO: 25 +23 
+ 
. . . QLRGGGGSGGGGSGGGGSQLVKSELEEKKSELRHKLK . . . 
SEQ ID NO: 26 +18.sup.3 
+ 
__________________________________________________________________________ 
.sup.1 The inserted aa residues are shown in boldtype. 
.sup.2 Activity based on the cleavage of pTZ19R DNA substrate. The 
cleavage pattern of the substrate by these mutants (as determined by the 
agarose gel electrophoresis) were similar to the wildtype (wt) FokI. 
.sup.3 The inserted aa residues contain (G--G--G--G--S).sub.3 linker. (SE 
ID NO:15) 
Analysis of Sequence-Specificity and the Cleavage Distances from the 
Recognition Site of the Mutant Enzymes 
The agarose gel electrophoretic profile of the products of pTZ19R substrate 
cleavage by FokI and the deletion and insertion mutants are shown in FIG. 
4. The profiles are very similar, suggesting that deletion and insertions 
ranging from -4 to +23 residues do not disrupt the sequence-specificity of 
the enzymes. Several clones of seven codon deletion mutants were 
identified; however, none of these clones showed any enzymatic activity 
indicating that seven residue (KSELEEK) (SEQ ID NO:14) deletion probably 
inactivates the enzyme. All digestions were done at similar protein 
concentrations. Larger insertion mutants show partial digests. These 
reactions proceed to completion either by increasing enzyme concentration 
or by digesting for longer time periods. 
To determine the distance of cleavage by the insertion and deletion mutants 
from the recognition site, the cleavage products of the .sup.32 P-labeled 
DNA substrates containing a single FokI site were analyzed by PAGE (FIGS. 
5 and 6). The digestion products were analyzed alongside the Maxam-Gilbert 
(G+A) sequencing reactions (19) of the substrates. The cut sites of the 
insertion mutants are all shifted one bp away from the recognition site on 
both strands of the DNA substrates as compared to the wild-type enzyme. A 
small amount of cleavage similar to that of wild-type enzyme is also 
observed. It is more pronounced with the four codon insertion (TAEL) (SEQ 
ID NO:4) mutant. Relaxation of specificity at the cut site is much more 
prevalent on the 5'-CATCC-3' strand than the 5'-GGATG-3 strand in the case 
of the insertion mutants. A similar relaxation of specificity at the cut 
site was observed with the "chimeric" restriction endonuclease produced by 
linking Ubx homeo domain to the cleavage domain of FokI (15). The four 
codon deletion (KSEL) (SEQ ID NO:3) mutant shows only relaxation of 
specificity at the cut site. The cleavage occurs predominantly at the site 
similar to the wild-type enzyme. The cut site is not shifted one bp closer 
to the recognition site as expected. 
There appears to be no simple relationship between the length of the 
protein connector region of FokI and the cleavage distance from the 
recognition site within its DNA substrate. The recognition and cleavage 
domains of FokI are likely held together by a non-structured loop. There 
is probably some association between the recognition and nuclease domains 
of FokI. This domain--domain interaction is likely to be weak since mixing 
of the purified FokI recognition domain with the nuclease domain does not 
reconstitute FokI endonuclease. No sequence-specific cleavage of the 
substrate is observed with such a mixture; only non-specific nuclease 
activity is observed. Furthermore, the 18 amino acid residue insertion 
mutant that includes the glycine linker, (Gly.sub.4 Ser).sub.3 (SEQ ID 
NO:15), also shows the same sequence-specificity as the wild-type enzyme 
(FIG. 4, lane 10); it also cleaves predominantly one nucleotide further 
away from the recognition site on both strands of the DNA substrate (FIGS. 
5 and 6; lane 9). 
The glycine linker should neither exhibit a propensity for ordered 
secondary structure (20), nor show any tendency to interfere with the 
folding of the individual domains of the mutant enzyme. It is also 
unlikely to interfere with the domain-domain interaction that occurs due 
to the association of the two domains. This protein-protein interaction 
between the domains probably leads to the cleavage of the substrate at a 
precise distance from the recognition site by the mutant enzyme. This may 
also explain the absence of a linear relationship between the length of 
the linker region of FokI and cleavage distance from the recognition site 
within the DNA substrate. 
The modular structure of FokI restriction endonuclease suggested that it 
may be feasible to engineer "chimeric" endonucleases with novel 
sequence-specificities by linking other DNA-binding domains to the 
cleavage domain of FokI endonuclease. Recently, the first chimeric 
restriction endonuclease has been successfully engineered by linking the 
Ubx homeo domain to the cleavage domain (F.sub.N) of FokI (15). 
In this regard, the present invention indicates that large insertions 
between the DNA-recognition domain and the catalytic domain of FokI do not 
disrupt the activity of the enzyme. Internal deletions of seven or more 
codons of the linker region appear to result in inactivation of the 
enzyme. These results are important for engineering "chimeric" restriction 
enzymes especially when one encounters protein-folding problems with the 
fusions. 
In addition, "artificial" restriction enzymes with tailor-made 
sequence-specificities may be designed that would be used in the mapping 
and sequencing of large genomes. Thus, an array of artificial nucleases 
with designed specificities may be engineered for various applications. 
More specifically, the mutants of the present invention have been designed 
for the same DNA sequence-specificity as the wild-type enzyme. However, 
the insertion mutants cleave predominantly one nucleotide further away 
from the recognition site on both strands of the DNA substrate. Thus, this 
invention provides new restriction enzymes with new cleavage sites 
compared to that of the wild type enzyme. Furthermore, the four codon 
deletion also provides a new restriction enzyme which exhibits relaxed 
specificity at the cut site while the seven codon deletion appears to 
inactivate the enzyme. 
These engineered endonucleases will greatly facilitate the manipulation and 
mapping of genomic DNA and will be used to obtain information about 
protein structure and design. 
Cleavage of Hemi-Methylated DNA Substrates by the Four Codon Insertion 
Mutants 
Recently, Waugh and Sauer have identified a novel class of FokI restriction 
endonuclease mutants that cleave hemi-methylated substrates (14). To test 
if the deletion and insertion mutants of FokI cleave hemi-methylated DNA 
sites, the same four oligonucleotides described by Waugh and Sauer were 
used as substrates (SEQ ID NO:12, SEQ ID NO:13, hemi-methylated 
oligonucleotide of SEQ ID NO:12 and hemi-methylated oligonucleotide of SEQ 
ID NO:13). By annealing various combination of the .sup.32 P-labelled 
oligonucleotides, an unmethylated substrate, two hemi-methylated 
substrates and the fully methylated substrate were obtained. Cleavage 
assays with the hemi-methylated substrates were performed with the 
deletion and insertion mutants of FokI endonuclease. Of these, only the 
four codon insertion, TAEL (SEQ ID NO:4) and KSEL (SEQ ID NO:3), mutants 
cleave hemi-methylated substrates (FIG. 7A, 7B, 7C and 7D). Both the 
wild-type and the four codon insertion mutants cleave unmethylated 
substrate to yield two fragments, and none of the enzymes cleave the 
doubly methylated substrate. In addition, the two mutant enzymes and not 
the wild-type enzyme cleave both forms of hemi-methylated DNA. Although 
the cleavage of hemi-methylated substrates is not as efficient as the 
unmethylated substrate, it proceeds reasonably well. The TAEL (SEQ ID 
NO:4) insertion mutant appears to cleave the hemi- methylated substrates 
better than the KSEL (SEQ ID NO:3) insertion mutant. 
Why does the four amino-acid residue insertions enable the mutant enzymes 
to cleave hemi-methylated DNA and not the larger inserts? The four codon 
insertions could increase the affinity of the mutants for the 
hemi-methylated DNA. The binding affinities of wild-type enzyme and both 
four codon insertion mutants were compared by filter binding assays using 
the synthetic oligonucleotide substrates (FIGS. 9A, 9B and 9C). The 
binding affinities were measured in the presence of non-specific DNA, poly 
dI-dC. The results suggest wild-type FokI can bind to hemi-methylated and 
even fully-methylated sites, although .about.100 fold less efficiently 
than to unmethylated sites. The TAEL (SEQ ID NO:4) and KSEL (SEQ ID NO:3) 
insertion mutants show similar results suggesting that insertions do not 
affect the binding step. Furthermore, this model does not account for the 
inability of the larger insertion mutants to cleave hemi-methylated FokI 
sites. 
In a more plausible model, the rate-limiting step of FokI cleavage reaction 
could involve the dissociation of the nuclease domain from the 
DNA-recognition domain. The methyl groups may inhibit the dissociation of 
the nuclease domain from the DNA-recognition domain through hydrophobic 
interactions or even stearic effects. The four amino acid residue 
insertions may have partly uncoupled the nuclease domain from the 
recognition domain resulting in the cleavage of the hemi-methylated 
substrates. Due to the added flexibility associated with large insertions, 
these may not uncouple the FokI nuclease domain from the DNA-binding 
domain. The model is consistent with the observation that "chimeric" 
restriction endonucleases obtained by linking other DNA-binding proteins 
to the nuclease domain of FokI exhibit not only sequence-specific cleavage 
that is determined by the DNA-binding protein but also non-specific 
nuclease activity which can be controlled by lowering the concentration of 
MgCl.sub.2 (15). The dissociation of the enzyme from the cleaved product 
as a rate-limiting step in the FokI cleavage reaction could not be ruled 
out at this time. 
In summary, the present results suggest that large insertions between the 
DNA-recognition domain and the catalytic domain of FokI do not disrupt the 
activity of the enzyme. Internal deletions of seven or more codons of the 
linker region appears to result in the inactivation of the enzyme. These 
findings are of importance for future engineering of "chimeric" 
restriction enzymes especially when one encounters protein-folding 
problems with the fusions. Several laboratories are in the process of 
determining the crystal structures of FokI and FokI-DNA complexes. These 
studies will provide detailed information about the mechanism of the FokI 
cleavage reaction and the domain-domain interactions within the 
protein-DNA complex at atomic resolution. 
While the invention has been described in connection with what is presently 
considered to be the most practical and preferred embodiment, it is to be 
understood that the invention is not limited to the disclosed embodiment, 
but on the contrary is intended to cover various modifications and 
equivalent arrangements included within the spirit and scope of the 
appended claims. 
Thus, it is to be understood that variations in the present invention can 
be made without departing from the novel aspects of this invention as 
defined in the claims. 
The following scientific article have been cited throughout the present 
application and are hereby incorporated by reference in their entirety and 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 26 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGCTCTAGACGGCGGTGGAGGATCAGGGGGAGGAGGTAGC40 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGACTAGTTGTGATCCGCCTCCGCCGCTACCTCCTCCCCC40 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LysSerGluLeu 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ThrA laGluLeu 
1 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ThrAlaGluLeuThrAlaGluLeu 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ThrAlaGluLeuThrAlaGluLeuThrAla GluLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
Thr AlaGluLeuThrAlaGluLeuLysSerGluLeuLysSerGluLeu 
151015 
GluGluLys 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ThrAlaGluLeuThrAlaGluLeuThrAlaGluLeuLysSerGluLeu 
1510 15 
LysSerGluLeuGluGluLys 
20 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ArgGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer 
151015 
GlnLeu 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CTAGAGTCAGATAGCGAAGACTTCGGGGATGGGCTTAATGGCCTTAGTTCACAA54 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AGCTTTGTGAACTAAGGCCATTAAGCCCATCCCCGAAGTCTTCGCTATCTGACT54 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CTAGAGTCAGAATTCGAAGACTTGCCGGATGATCTGCAGGCCAGCTGTGGCGTCTAAATT60 
GA 62 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AGCTTCAATTTAGACGCCACAGCTGGCCTGCAGATCATCCGGCAAGTCTTCGAATTCTGA60 
CT62 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LysSerGluLeuGluGluLys 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer 
1510 15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GlyAlaAlaAsnAsnAsnAsnAsnAsnAr gThrGlyGly 
1510 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GlyAlaAlaAsnAsnAsnAsnAsnAsnAsnArgThrGlyGly 
1510 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GlnLeuValLysSerGluLeuArgHisLysLeuLys 
1510 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
( B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GlnLeuValGluGluLysLysSerGluLeuArgHisLysLeuLys 
1510 15 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GlnLeuValLysSerGluLeuGluGluLysLysSerGl uLeuArgHis 
151015 
LysLeuLys 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GlnLeuValThrAlaGluLeuLysSerGluLeuGluGluLysLysSer 
151015 
GluLeuArgHisLys LeuLys 
20 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GlnLeuValThrAlaGluLeu ThrAlaGluLeuLysSerGluLeuGlu 
151015 
GluLysLysSerGluLeuArgHisLysLeuLys 
2025 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GlnLeuValThrAlaGluLeuThrAlaGluLeuThrAlaGluLeuLy s 
151015 
SerGluLeuGluGluLysLysSerGluLeuArgHisLysLeuLys 
202530 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GlnLeuValThrAlaGluLeuThrAlaGluLeuLysSerGluLeu Lys 
151015 
SerGluLeuGluGluLysLysSerGluLeuGluGluLysLysSerGlu 
2025 30 
LeuArgHisLysLeuLys 
35 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
Gln LeuValThrAlaGluLeuThrAlaGluLeuThrAlaGluLeuLys 
151015 
SerGluLeuLysSerGluLeuGluGluLysLysSerGluLeuGluGlu 
202530 
LysLysSerGluLeuArgHisLysLeuLys 
3540 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: PEPTIDE 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
GlnLeuArgGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGly 
1510 15 
GlySerGlnLeuValLysSerGluLeuGluGluLysLysSerGluLeu 
202530 
ArgHisLysLeuLys 
35