Nucleic Acids Research, 2001, Vol. 29, No. 24 e120
© 2001 Oxford University Press
FspAI, a unique type II restriction endonuclease that recognizes the octanucleotide sequence 5'-RTGC
GCAY-3'
MBI Fermentas, and 1Institute of Biotechnology, Graiciuno 8, 2028 Vilnius, Lithuania
Received August 30, 2001; Revised and Accepted October 8, 2001.
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ABSTRACT |
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A new type II restriction endonuclease designated FspAI has been partially purified from a Flexibacter species Tv-m21K. FspAI recognizes the octanucleotide sequence 5'-RTGC
GCAY-3' and cleaves it in the center generating blunt-ended DNA fragments. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More than 3000 type II restriction endonucleases have been identified to date (1). The majority of them recognize 4–6 bp nucleotide sequences. Thus far, only 25 enzymes are known, whose recognition specificities require 8 nt. They represent 12 different nucleotide sequences, among which true palindroms (2–7), interrupted palindroms (8) or palindroms with degenerate positions (9) have been found. Rare cutting endonucleases are very useful for the mapping of large genomes and, therefore, further expansion of their repertoire is desirable. Here we describe the isolation and characterization of the restriction endonuclease FspAI from Flexibacter species Tv-m21K, which recognizes the octanucleotide sequence 5'-RTGC
GCAY-3'. |
MATERIALS AND METHODS |
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Biological materials
Flexibacter species strain Tv-m21K was obtained from MBI Fermentas. The cells were grown aerobically at 28°C in a medium containing 10 g/l peptone, 5 g/l Lab-lemco powder (both from Oxoid), 5 g/l NaCl and 0.25 g/l MgSO4.7H2O, pH 7.8, up to late-logarithmic phase, collected by centrifugation and stored at –20°C until use. Bacteriophage
, plasmid DNA, Cycle Reader DNA Sequencing kit, DNA size markers and all restriction endonucleases used were products of MBI Fermentas. SssI methylase was purchased from New England Biolabs, Ad2 DNA from Sigma and [
-33P]dATP from Amersham.
Endonuclease assay
The restriction endonuclease activity was assayed in 50 µl of MBI Fermentas O+ buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 0.1 mg/ml BSA). Column samples (2 µl) were added to the reaction mixture, containing 1 µg phage DNA-Psp1406I fragments, incubated for 15 min at 37°C and an aliquot from each solution was then electrophoresed in a 0.7% agarose gel.
Purification of the restriction endonuclease
All steps were carried out at 4°C. Frozen cells (25 g wet weight) were thawed in 75 ml buffer A (10 mM K-phosphate, pH 7.0, 1 mM EDTA, 7 mM 2-mercaptoethanol), containing 0.3 M NaCl. After sonication, insoluble material was removed from the crude extract by centrifugation at 30 000 g for 1 h.
The crude extract was applied to a heparin–Sepharose column (1.5 x 17 cm) pre-equilibrated with buffer A, 0.3 M NaCl. The column was washed with the same buffer and eluted with a 450 ml linear gradient from 0.3 to 1.3 M NaCl. Fractions of 10 ml were collected and assayed for endonuclease activity. The restriction endonuclease FspAI eluted at 
0.83–0.94 M NaCl. Active fractions were pooled and dialyzed against buffer A containing 0.3 M NaCl.
The enzyme pool from heparin–Sepharose was applied to an AH–Sepharose column (1.5 x 13 cm) equilibrated with the dialysis buffer. The column was washed with the same buffer and eluted with a 300 ml linear gradient from 0.3 to 1.3 M NaCl in buffer A. Fractions of 6 ml were collected. FspAI activity eluted from the column at 0.55–0.72 M NaCl. These fractions were pooled and dialyzed against buffer A containing 0.3 M NaCl.
Active fractions from AH–Sepharose after dialysis were applied to a phosphocellulose P11 column (1.0 x 16 cm), equilibrated with buffer A containing 0.3 M NaCl. The column was washed with the same buffer and subsequently developed with a 200 ml linear gradient from 0.3 to 1.3 M NaCl in buffer A. Fractions of 4 ml were collected. The peak of the restriction endonuclease activity eluted at 0.78–0.94 M NaCl. Active fractions were pooled and dialyzed against 10 mM Tris–HCl pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 50% glycerol. The final preparation was stored at –20°C. The yield of the enzyme was 1500 U/g wet weight of cells. (One unit of enzyme is the amount required to hydrolyze 1 µg of DNA in 60 min in a total reaction volume of 50 µl.)
Determination of the recognition sequence and cleavage site
The recognition sequence of FspAI was inferred by restriction mapping of the recognition sites on the DNAs of phage , adenovirus-2 and plasmid pBR322. Then the fragments predicted by cleavage of the inferred recognition sites were compared with the observed restriction fragments from the FspAI cleavage of the DNAs.
DNA was used as a template to characterize the cleavage site of FspAI. A 20mer oligodeoxyribonucleotide complementary to between positions 21 720 and 21 740 was used in direct sequencing through the FspAI site located at position 21 804. Four dideoxy sequencing reactions using [-33P]dATP and Cycle Reader DNA Sequencing kit were carried out. The same primer and template were used in an extension reaction, which also included T7 DNA polymerase, dNTP and [-33P]dATP. The extension reaction was heat inactivated, treated with FspAI and the reaction mixture was divided into two halves. One sample was treated with T4 DNA polymerase. Both samples were diluted with sequencing dye solution and loaded on a standard sequencing gel along with the sequencing reactions.
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RESULTS |
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The recognition sequence and cleavage site of FspAI
To determine the substrate specificity of the enzyme, several DNA substrates (
, T7, Ad2, pBR322, pUC19, 
X174 and M13mp18; 1 µg each) were incubated with FspAI in 50 µl of MBI Fermentas O+ buffer at 37°C. FspAI did not cleave X174, M13mp18 or pUC19 DNAs. Two FspAI cleavage sites were obtained with pBR322 DNA, which were mapped to approximate positions 260 and 1450 using HindIII, BamHI, PaeI, Bsp68I, Mva1269I, Kpn2I, NdeI, CaiI and PstI. Restriction endonucleases Acc65I, PdiI, SacI, Psp5II and XhoI were used to map an FspAI cleavage site in DNA (approximate position 21 800). A computer-aided analysis of the mapped FspAI sites and their vicinity revealed a single common sequence, 5'-RTGCGCAY-3'. This sequence appears twice in DNA at positions 21 804 and 21 825. Consistent with this prediction, only two fragments of 21 800 and 26 700 bp were detected after electrophoresis of digestion products of this DNA with FspAI in an agarose gel (data not shown), the third fragment was too short (21 bp) to be detected by this method. The number and sizes of DNA fragments generated by FspAI digestion of adenovirus-2 (six fragments of 12 200, 11 600, 6000, 4500, 1300 and 370 bp), T7 (three fragments of 30 000, 11 100 and 3400 bp) and pBR322 (two fragments of 3160 and 1200 bp) DNA were consistent with the predicted recognition sequence (Fig. 1A). Additional evidence supporting this conclusion was obtained after the double digestion of pBR322 DNA with FspAI and NsbI (Fig. 1B). As all FspAI sites are also recognized by NsbI (recognition sequence 5'-TGCGCA-3'), double digestion of any DNA substrate with these enzymes should therefore yield the NsbI pattern. Data presented in Figure 1B show that this was the case.
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The cleavage position of FspAI was determined by comparison of dideoxy sequencing ladders with fragments generated by FspAI cleavage and T4 DNA polymerase action on the digestion product. The results of the determination of the FspAI cleavage site are shown in Figure 2. The fragment generated by FspAI digestion co-migrates with the first C-band of the sequence ladder (Fig. 2, lane 1) in the recognition site. From these results it can be inferred that FspAI cleaves DNA in the center of the recognition sequence 5'-ATGC
GCAT-3'. Lane 2 shows the result when the fragment produced by FspAI was further treated with T4 DNA polymerase. The single band obtained after such treatment also co-migrates with the C-band of the sequence ladder confirming that the cleavage point on the complementary DNA strand is the same. These results thus unequivocally indicate that FspAI cleaves its target site in the middle on both strands 5'-RTGCGCAY-3' generating fragments with blunt ends.
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Enzymatic properties
FspAI shows the highest activity in high ionic strength buffers. Optimal enzyme activity is observed in 50 mM Tris–HCl buffer pH 7.5, containing 10 mM MgCl2 and 100 mM NaCl. Incubation temperature does not considerably influence enzyme activity in the range of 20–37°C.
The recognition sequence of FspAI includes the CpG dinucleotide, which is a common methylation site in eukaryotic DNA. Therefore, the sensitivity to cytosine-5 methylation in the recognition site was investigated. After methylation of and pBR322 DNA with the SssI methylase, both substrates became totally resistant to FspAI cleavage (data not shown), indicating that FspAI is inhibited by CpG methylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Restriction endonucleases that recognize sequences longer than 6 bp have found great use in the mapping of complex DNA molecules like chromosomes. FspAI, which recognizes the 5'-RTGCGCAY-3' octanucleotide sequence, is a new addition to the list of such valuable tools for the mapping of large genomes and for the cloning of high molecular weight DNA fragments. Computer analysis indicates that FspAI should cleave Escherichia coli and Saccharomyces cerevisiae genomes into fragments of an average length of 11 and 52 kb, respectively. The calculated average fragment size of the nineteenth human chromosome (55 500 bp) is almost twice as low as that for NotI (99 500 bp) but considerably higher than for SfiI (26 300 bp). Given that FspAI is sensitive to CpG methylation, the cleavage frequency of human or other eukaryotic DNA may be much lower than that calculated for unmodified DNA.
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ACKNOWLEDGEMENT |
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The authors would like to thank Dr Saulius Klimaauskas for help in preparation of the manuscript.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +370 2 602110; Fax: +370 2 602116; Email: janulait{at}ibt.lt
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REFERENCES |
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