Gene, 172 (1996) 49-51

0 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00 49

GENE 09579

BstFSI, an unusual isoschizomer of FokI

(Restriction endonuclease; recognition sequence; cleavage points)

M.A. Abdurashitov, E.V. Kileva, N.M. Shinkarenko, A.V. Shevchenko, V.S. Dedkov and
S.Kh. Degtyarev

SibEnzyme, Novosibirsk 90, 630090, Russia

Received by F. Banany: 12 July 1995; Revised/Accepted: 26 July/l3 September 1995; Received at publishers: 14 December 1995

SUMMARY

BstFSI, a new restriction endonuclease (ENase) from Bacillus stearothermophilus F5, has been discovered. This enzyme
recognizes S-GGATG-3’ and cleaves DNA, generating a 2-base 3’ extension:
5’ -GGATG NN"-3'
3' -CCTAC,NN -5'
BstFSI is an isoschizomer of FokI and seems to be evolutionarily close to other nonpalindromic-recognizing ENases
from thermophilic bacilli.

INTRODUCTION GGATG( 10/14), respectively (Sugisaki and Kanazawa,

1981; Kita et al., 1992). We report here a new FokI isosch-
Type-II ENases cleave DNA containing their recogni- izomer BstF51 which recognizes the same sequence but
tion sequences in a highly specific manner. As a rule, cleaves DNA at GGATG(2/0). This is a first example of
ENases with the same specificity isolated from different class-IIS heteroschizomeric ‘trio’.
sources cut both DNA strands at the same positions.
However, some ENases recognizing the same DNA
sequence cleave it at different nucleotides. For example, EXPERIMENTAL AND DISCUSSION
BbeI, EheI, KasI and NurI, all recognize GGCGCC but
cut DNA in a four different manners (Roberts and (a) Isolation and determination of recognition sequence
Macelis, 1993). Nevertheless, all cleavage positions of B. stearothermophilus F5 was grown at 50°C in a
these enzymes are located within this recognition medium containing (per liter) 10 g peptone/ g yeast
sequence. extract/l g NaCl (pH 7.5 at 25°C). Cells were disrupted
There are of isoschizomers that cleave DNA outside by sonication.
the recognition sequence in a different manner. One of BstF51 ENase was isolated by phosphocellulose,
them is FokI and StsI ENases which were shown to recog- DEAE-cellulose and heparin-sepharose chromatograph-
nize GGATG but cleave DNA at GGATG(9/13) and ies using 0.2-l M KC1 linear gradient in 20 mM
Tris-HCl elution buffer (pH 7.5). The optimal conditions
Correspondence to: Dr. M. A. Abdurashitov, SibEnzyme, 8 Lavrentiev
Str., Novosibirsk-90, 630090, Russia. Tel. (7-383) 235-3350; Fax (7-383) for purified enzyme activity were 20 mM Tris - HCl
232-8831; e-mail:info@siben.nsk.su pH 8.5/10 mM MgCl,/lOO mM NaCl at 55°C.
The lengths of fragments produced by BstF51 on com-
Abbreviations: aa, amino acid(s); Ad, adenovirus; B. , Bacillus; ENase,
monly used viral and plasmid DNAs (Fig. 1) correspond
restriction endonuclease; nt, nucleotide(s); oligo, oligodeoxyribonucleo-
tide(s); PAGE, polyacrylamide gel electrophoresis; PolIk, Klenow to those predicted for FokI ENase. This is an evidence of
(large) fragment of E. coli DNA polymerase I. the both enzymes recognition sequences equivalency.

PII 0378-1119(96)00190-4
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1 2 3 4 5 6 7
a EACGTK
I .
PUI 3’

b
3’
A G AEE

Fig. 1. Digest of phage h (lane 2), T7 (lane 3), Ad2 (lane 4), pBR322
(lane 5) and pUC19 (lane 6) DNAs with BstF5I. DNA fragment lengths
marker: digest of phage h DNA with Bmel8I (isoschizomer of AvaII)
(lanes 1 and 7). Reaction products were separated on a 1% agarose gel.

(b) Determination of cleavage points

To determine the DNA cleavage points of the new
enzyme, two modifications of dideoxynucleotide chain-
termination method (Brown and Smith, 1980) have been
employed (Fig. 2). The comparison of resulting bands
with sequencing lanes indicated that the new ENase
cleaves double stranded DNA as it is shown below:
5 '-GGATG NN"-3'
3'-CCTAC,NN -5'.
Fig. 2. BstFSI cleavage points determination. (a) Linearized pUC19
So, BstF51 recognizes 5-bp sequence which is identical DNA was used as the template for Taq DNA polymerase reaction
to the sequence recognized by FokI and StsI ENases. starting with y-32P-labelled reverse lh-mer primer, which was comple-
However, all three enzymes have different cleavage posi- mentary to 461-476 region of the plasmid. The primed-synthesis reac-
tion products were cut using BstF51 and half of the sample was treated
tions (2/O, 9/13 and 10/14, respectively, using standard
with PolIk. (b) Phage T7 DNA region containing two near-by opposite-
brief designations). Taking into account the cleavage oriented BstF51 sites was the template and 1%mer y-32P-labelled reverse
manner and recognition site similarity (Table I) BstFSI primer complementary to nt 15 792-15 809 region of T7 was extended
may be evolutionarily relative to other ENases found in by Taq polymerase and the reaction products were cut with BstF51.
B. stearothermophilus strains rather than to the prototype. Lanes A, C, G, T, products of the standard polymerase sequencing
reactions with addition of the corresponding dideoxynucleotides; lanes
E, polymerase reaction products cut with BstFSI; lane K, as in E with
further treatment with PolIk. Products were separated by a 7 M
TABLE I urea-6% PAGE.
Comparison of recognition sequences of four ENases from
B. stearothermophilus strains
(c) Possible applications
ENase Recognition sequence The new enzyme may have many applications in
molecular biology. One of the possible applications of
Upper strand Bottom strand
BstFSI is DNA cleavage after FokI or StsI treatment. A
BstFS 1 5’-GGATGNN”-3’ 5’-N,CATCC-3’ short oligo produced by this manner near the FokI
BsrDI 5’-GCAATGNN”-3’ 5’-N,CATTGC-3’ recognition sequence may be replaced by synthetic oligo
BS?d 5’-GAATGCN”-3’ 5’-G,CATTC-3’
of interest. Subsequent DNA ligation allows to replace
BsrI 5’-ACTGGN”-3’ 5’-C,CAGT-3’
7 nt resulting in a change of l-3 aa in corresponding
 51

protein. A particular feature of F&I and other class-IIS Kita, K., Kotani, H., Ohta, H., Yanase, H. and Kato, N.: StsI, a new
FokI isoschizomer from Streptococcus sanguis 54, cleaves
restriction endonucleases is a possibility of DNA-pro-
S’GGATG(N) 1,,,143’. Nucleic Acids Res. 20 (1992) 618.
ducts ligation with formation of original molecule Roberts, R.J. and Macelis, D.: Restriction endonucleases and their
(Szybalski et al., 1991). FokI+BstFSI double digest of isoschizomers. Nucleic Acids Res. 21 (1993) 3125-3137.
DNA does not break this property and it creates a new Sugisaki, H. and Kanazawa, S.: New restriction endonucleases from
and simple opportunity to carry out a DNA mutagenesis. Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MU).
Gene 16 (1981) 73-78.
Szybalski, W., Kim, S.C., Hasan, N. and Podhajska, A.: Class-IIS
restriction enzymes - a review. Gene 100 (1991) l-39.
REFERENCES

Brown, N.L. and Smith, M.: A general method for defining restriction
enzyme cleavage and recognition sites. Methods Enzymol. 65
(1980) 391-404.