BstAPI, an ApaBI isoschizomer, cleaves DNA at 5[prime]-GCANNNN[darr]NTGC-3[prime]
BstAPI, an ApaBI isoschizomer, cleaves DNA at 5[prime]-GCANNNN[darr]NTGC-3[prime]
Murat A. Abdurashitov*, Olga A. Belichenko, Alla V. Shevchenko, Vladimir S. Dedkov, Sergey Kh. Degtyarev
SibEnzyme, 8 Lavrentiev Str., Novosibirsk 630090, Russia
Received March 21, 1997;Revised and Accepted May 1, 1997
ABSTRACT
Cleavage positions of BstAPI, a new restriction endonuclease (ENase) that recognizes palindromic interrupted DNA sequence, have been determined. Recognition sequences and cleavage sites comparison shows that BstAPI shares similarity with a number of type II restriction enzymes.
BstAPI, a new restriction ENase from Bacillus stearothermophilus AP has been isolated using phosphocellulose, hydroxyapatite and heparin-sepharose chromatographic steps. Electrophoretical patterns of BstAPI cleavage products for different DNAs (Fig. 1) correspond to those predicted for ApaBI restriction ENase that recognizes interrupted DNA sequence 5[prime]-GCAN5TGC-3[prime] (1). Optimal conditions for BstAPI activity are 10 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 100 mM NaCl and 1 mM dithiotreitol at 60°C.
The cleavage site of the new enzyme has been determined by modified dideoxynucleotide chain-termination method (2). The data obtained (Fig. 2) indicate that BstAPI cleaves DNA sequence as it is shown by arrow: 5[prime]-GCANNNN[darr]NTGC-3[prime]. This result has been confirmed by cleavage of a 33 bp labelled synthetic oligodeoxyribonucleotide duplex with BstAPI and some other restriction ENases (Fig. 3).
Figure 1BstAPI digests on different substrate DNAs. Lane 1, lambda; lane 2, T7; lane 3, pBR322; lane 4, pBR322 linearized with ScaI. Lanes M, size standard - [lambda]DNA cleaved with Bme18I(isoschizomer of AvaII).
Figure 2 Determination of BstAPI cleavage position by sequencing through it recognition site at position 2291 on pBR322 DNA. Lanes A, C, G, T are standard sequencing lanes; lane R, primer extension products cleaved with BstAPI.
Figure 3 Enzymatic digests of oligonucleotide duplex containing 32P-labelled strand. Lane 1, uncleaved duplex; lane 2, VneI(recognition sequence G[darr]TGCAC); lane 3, BstAPI;lane 4, BstAPI+BstBAI (YAC[darr]GTR); lane 5, BstBAI; lane 6, Zsp2I(ATGCA[darr]T).
Thus, the cleavage site of BstAPI differs from that reported for ApaBI (5[prime]-GCANNNNN[darr]TGC-3[prime]). However, it correlates to the cleavage position of six other restriction ENases which recognize the DNA sequences slightly differing from BstAPI recognition site (Table 1). All these enzymes produce 3[prime]-protruding DNA fragments with three nucleotides sticky ends and contain cytosine in a second position of a general recognition sequence. One can suggest that a mechanism of DNA cleavage by all these enzymes is similar. Earlier we have observed the same situation with a group of restriction enzymes with non-palindromic recognition sequences (3). We believe that there are several general schemes of the DNA cleavage by restriction ENases and these schemes are tightly connected with a definite structure of recognition sites.
. Comparison of BstAPI cleavage site with those of the ENases with the most similar recognition sequences
ENase
Recognition sequencea
1
2
3
4
5
6
7
8
9
10
11
BstAPI
5[prime]-
G
C
A
N
N
N
N[darr]
N
T
G
C
-3[prime]
BglI
5[prime]-
G
C
c
N
N
N
N[darr]
N
g
G
C
-3[prime]
SfiI
5[prime]-g
G
C
c
N
N
N
N[darr]
N
g
G
C
c-3[prime]
MwoI
5[prime]-
G
C
n
N
N
N
N[darr]
N
n
G
C
-3[prime]
PflMI
5[prime]-
c
C
A
N
N
N
N[darr]
N
T
G
g
-3[prime]
DraIII
5[prime]-
C
A
c
N
N
N[darr]
g
T
G
-3[prime]
AlwNI
5[prime]-
C
A
g
N
N
N[darr]
c
T
G
-3[prime]
Recognition sequences are taken from REBASE database (4).aNucleotides that differ from correspondent nucleotides in BstAPI recognition sequence (or are additional to it) are in lowercase.
ACKNOWLEDGEMENT
The authors thank Danila Gonchar for critical reading of this manuscript.
REFERENCES
1. Grones,J. and Turna,J. (1993) Biochim. Biophys. Acta1162,323-325.
2. Brown,N.L. and Smith,M. (1980) Methods Enzymol.65, 391-404.MEDLINE Abstract
S. Kh. Degtyarev, O. A. Belichenko, N. A. Lebedeva, V. S. Dedkov, and M. A. Abdurashitov BtrI, a novel restriction endonuclease, recognises the non-palindromic sequence 5'-CACGTC(-3/-3)-3'
Nucleic Acids Res.,
June 1, 2000;
28(11):
e56 - e56.
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