Nucleic Acids Research, 2002, Vol. 30, No. 19 e98
© 2002 Oxford University Press
PfoI, a unique type II restriction endonuclease that recognises the sequence 5'-T
CCNGGA-3'
1
yt
Institute of Biotechnology and
1 Fermentas UAB, Grai
i
no 8, 2028 Vilnius, Lithuania
*To whom correspondence should be addressed. Tel: +370 2 602110; Fax: +370 2 602116; Email: janulait{at}ibt.lt
Received July 26, 2002; Accepted August 10, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T
CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Pseudomonas genus has proved to be a rich source of restriction endonucleases. To date, 136 type II enzymes produced by different strains belonging to this genus have been identified (REBASE version 207) (1). They recognise 58 different nucleotide sequences of 238 known. Most of them are represented by symmetric palindromes or partial palindromes. We describe here the isolation and characterisation of PfoI, a novel type II restriction endonuclease from Pseudomonas fluorescens biovar 126 which recognises an interrupted hexanucleotide sequence with two-fold rotational symmetry, 5'-T
CCNGGA-3', and cleaves it to generate 5 nt protruding 5'-ends. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biological materials
Pseudomonas fluorescens biovar 126 was from the Fermentas culture collection. The cells were grown aerobically at 30°C in a medium containing 10 g/l peptone, 5 g/l yeast extract (both from Oxoid), 10 g/l NaCl and 0.25 g/l MgSO4.7H2O, pH 7.3, up to late-logarithmic phase, collected by centrifugation and stored at –20°C until use. The phage
DNA, plasmid DNAs, Cycle Reader DNA Sequencing kit, DNA size markers and all restriction endonucleases used were products of Fermentas. SssI methylase was purchased from New England Biolabs, adenovirus 2 DNA from Invitrogen, T7 DNA from Sibenzyme and [
-33P]dATP from Amersham.
Endonuclease assay
The restriction endonuclease activity was assayed in 50 µl of Fermentas Y+ buffer (33 mM Tris–acetate pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate and 0.1 mg/ml BSA). Column samples (1 µl) were added to the reaction mixture, containing 1 µg phage DNA, incubated for 10 min at 37°C and an aliquot from each probe was then electrophoresed in a 0.7% agarose gel.
Purification of restriction endonuclease
All steps were carried out at 4°C. Frozen cells (100 g wet weight) were thawed in 200 ml of buffer A (10 mM potassium phosphate, pH 7.4, 1 mM EDTA, 7 mM 2-mercaptoethanol), containing 0.1 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 (2.6 x 20 cm) pre-equilibrated with buffer A containing 0.1 M NaCl. The column was washed with the same buffer and eluted with a 1000 ml linear gradient from 0.1 to 1.2 M NaCl. Fractions of 20 ml were collected and assayed for endonuclease activity. The restriction endonuclease PfoI eluted at 
0.76–0.88 M NaCl. Active fractions were pooled and dialysed against buffer A containing 0.1 M NaCl.
The enzyme pool from heparin–Sepharose was applied to a Q-Sepharose column (2.6 x 15 cm) equilibrated with the dialysis buffer. The column was washed with the same buffer and subsequently developed with a 750 ml linear gradient from 0.1 to 0.8 M NaCl in buffer A. Fractions of 15 ml were collected. PfoI activity eluted from the column at 0.30–0.35 M NaCl.
Active fractions from Q-Sepharose were applied to a hydroxyapatite column (1.6 x 21 cm), equilibrated with buffer A containing 0.1 M NaCl. The column was washed with the same buffer and eluted with a 400 ml linear gradient from 0.01 to 0.4 M potassium phosphate in buffer A. Fractions of 8 ml were collected. The peak of the restriction endonuclease activity eluted at 0.26–0.30 M potassium phosphate. Active fractions were pooled and dialysed against 10 mM Tris–HCl pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM DTT and 50% glycerol. The final preparation was stored at –20°C. The yield of the enzyme was 1500 U/g wet weight of cells (1 U of enzyme is the amount required to hydrolyse 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 PfoI 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 at the inferred recognition sites were compared with the observed restriction fragments from PfoI cleavage of various different DNAs. Phage DNA was used as a template to characterise the cleavage site of PfoI. A 20mer oligodeoxyribonucleotide complementary to DNA between positions 13 860 and 13 880 was used in direct sequencing through the PfoI site located at position 13917. Four dideoxy sequencing reactions (lanes G, A, T and C, respectively) using [-33P]dATP and a Cycle Reader DNA Sequencing kit (Fermentas) 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 and then the PfoI-digested radiolabelled DNA probe was divided into two. One sample was treated with T4 DNA polymerase. Both samples were diluted with sequencing dye solution and loaded on a standard sequencing gel together with the dideoxy sequencing reactions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recognition sequence and cleavage site of PfoI
To determine the substrate specificity of the enzyme, DNA substrates (
, T7, Ad2, pBR322, pUC19, 
X174 and M13mp18 DNAs) were incubated at 37°C in 50 µl of Fermentas Y+ buffer containing 1 µg of DNA. PfoI did not cleave X174, M13mp18 and T7 DNAs. The single PfoI cleavage site in pBR322 DNA was mapped to approximate position 2100 using HindIII, BamHI, Eco88I, Kpn2I, PvuII, CaiI, Eam1105I and PstI (data not shown). Eight cleavage sites on DNA were mapped (approximate positions 4000, 8800, 12 950, 14 500, 18 400, 22 350, 26 500 and 46 900) by double digestion with Acc65I, BplI, Bsp120I, Eco47III, Eco81I, Eco105I, EheI, FspAI, NheI, PdiI, Pfl23II, Psp5II, SacI and XhoI. A computer-aided search of homologous nucleotide sequences at the mapped PfoI sites and areas surrounding them revealed only one common sequence, 5'-TCC(C/G)GGA-3'. The predicted PfoI target site was consistent with the absence of PfoI sites in T7, X174 and M13mp18, the presence of a single PfoI cleavage site in pBR322 and pUC19 and the number and sizes of fragments generated by PfoI digestion of DNA (Fig. 1). However, the results with Ad2 DNA indicate that the recognition site of PfoI contains in the middle any nucleotide (N), not the C/G base pair. According to its published sequence (2), Ad2 DNA contains only one sequence 5'-TCC(C/G)GGA-3', at position 28 571, and nine 5'-TCC(A/T)GGA-3' sites. PfoI degraded this DNA into 11 fragments. The number and size of the fragments were fully in line with those predicted for sequence 5'-TCCNGGA-3'. The apparently conflicting results obtained with PfoI digests of other DNAs used (Fig. 1) can be explained by the absence of the sequence 5'-TCC(A/T)GGA-3' in these substrates. Thus, experimental data strongly suggest that PfoI recognises the sequence 5'-TCCNGGA-3'. No restriction endonuclease of such specificity has been known before.
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The cleavage point of PfoI was determined by comparison of dideoxy sequencing ladders with fragments generated by PfoI cleavage and T4 DNA polymerase action on the digestion product. Results of the determination of the PfoI cleavage site are shown in Figure 2. The fragment generated by PfoI digestion co-migrates with the T band of the sequence ladder (Fig. 2, lane 2) through the PfoI recognition site. From these results it can be inferred that PfoI cleaves DNA after the first nucleotide of the PfoI recognition sequence starting from the 5'-end as indicated: 5'-T
CCGGGA-3'. Lane 1 shows the result when the fragment produced by PfoI digestion was further treated with T4 DNA polymerase. The single band obtained by the polymerase activity of T4 DNA polymerase co-migrates with the G band of the sequence ladder TCCGGGA and indicates the cleavage point on the complementary DNA strand. These results would therefore indicate that PfoI cleaves double-stranded DNA generating fragments with 5 nt protruding 5'-ends and cleavage specificity is 5'-TCCNGGA-3'.
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Enzymatic properties
The restriction endonuclease PfoI is strictly dependent on Mg2+, but does not require S-adenosylmethionine or ATP for its activity. Optimal PfoI activity, as judged by the absence of partial digestion products at a constant enzyme to DNA ratio, was found to be at salt concentrations between 0.05 and 0.1 M. The most effective digestion was observed within the pH range 7.5–8.0. A considerable loss of endonuclease activity was observed at higher pH values, beginning from 8.5. The optimal temperature for cleavage was found to be 37°C.
Hydrolysis of one of the fifteen PfoI sites in phage DNA (position 22 349) was shown to be approximately 50 times slower in comparison with the other sites. Slow cleavage rates of certain sites have been observed with a variety of other restriction enzymes (3–6). The resistance to cleavage was attributed either to the recognition sequence itself (for enzymes recognising degenerate nucleotide sequences) due to the alternating guanosine and cytosine residues or to G+C-rich flanking sequences possibly related to kinked DNA structures. Examination of the sequence surrounding the resistant PfoI site showed a significantly higher G+C content in this region (5'-CCGGATCCCGGAGGCGG-3'). In addition, we found that the more resistant site partially overlaps the Dam methylation sequence, producing a hemimethylated PfoI site in DNA modified by Dam methylase. However, any effect of Dam methylation can be ruled out because the slow cleavage of this site is also observed with DNA isolated from Escherichia coli dam– mutant hosts. Moreover, this conclusion is supported by the observation that plasmid DNAs with PfoI sites overlapping Dam methylation sites are cleaved at a normal rate. Thus, a strongly reduced cleavage rate at one particular PfoI site in DNA could probably be attributed to the nature of the nucleotides flanking the recognition sequence.
Depending on the central nucleotide pair the PfoI recognition sequence may also overlap Dcm or CG methylation sites. Since such methylation may also affect DNA cleavage, enzyme sensitivity to them was investigated. After methylation of DNA with SssI methylase, the substrate became almost totally resistant to PfoI cleavage (data not shown), indicating that PfoI is inhibited by CpG methylation. The enzyme also did not cleave the pACYC177 plasmid DNA single PfoI site, which overlaps a Dcm methylase site. Therefore, it may be concluded that internal cytosine methylation may interfere with DNA cleavage by PfoI.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A new type II restriction endonuclease PfoI with the novel sequence specificity 5'-T
CCNGGA-3' has been discovered and purified from P.fluorescens biovar 126. Based on its characteristics (palindromic nucleotide sequence, cleavage within recognition sequence, Mg2+ as the only cofactor) PfoI can be classified as an orthodox type II restriction endonuclease. Its recognition sequence is a new addition to the family of hexanucleotide sequences interrupted by one non-specific nucleotide recognised by BstEII (5'-GGTNACC-3'), EspI (5'-GCTNAGC-3') and SauI (5'-CCTNAGG-3'). PfoI could be used for ligation of DNA fragments in conjunction with the following enzymes which are related in sequence specificity and cleavage position: EcoRII [CC(A/T)GG], BssKI (CCNGG), EcoHI [CC(C/G)GG] and SexAI [ACC(A/T)GGT]. PfoI is inhibited by C-5 methylation at the internal cytosine residues of its recognition sequence: 5'-TCm5CNGGA-3'. Thus, PfoI cleavage at 5'-TCm5CWGGA-3' sites is prevented on DNA isolated from E.coli strains containing Dcm methylase. As PfoI cleavage at 5'-TCm5CSGGA-3' sites is not inhibited by this methylation, selective methylation of 5'-TCCWGGA-3' sequences may be used to restrict PfoI sequence specificity to 5'-TCCSGGA-3'. A restriction enzyme of such specificity has not been found so far. If complete PfoI digestion is required, DNA must be isolated from E.coli dcm– cells. When working with eukaryote DNAs, cleavage by PfoI may be also affected by CG methylation. DNA methylated at CG sites (using SssI methyltransferase) may be cleaved by PfoI only at 5'-TCCWGGA-3' sites.
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REFERENCES |
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[Abstract/Free Full Text] - Oller,A.R., Vanden Broek,W., Conrad,M. and Topal,M.D. (1991) Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. Biochemistry, 30, 2543–2549.[Medline]
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