| Nucleic Acids Research | Pages |
BseSI, a restriction endonuclease from Bacillus stearothermophilus Jo 10-553, which recognizes the novel hexanucleotide sequence 5[prime]-G(G/T)GC(A/C)[darr]C-3[prime]
Introduction
Methods And Results
Discussion
Acknowledgements
References
BseSI, a restriction endonuclease from Bacillus stearothermophilus Jo 10-553, which recognizes the novel hexanucleotide sequence 5[prime]-G(G/T)GC(A/C)[darr]C-3[prime]
Received March 22, 1999; Revised and Accepted May 10, 1999
ABSTRACT A new restriction endonuclease BseSI has been isolated from Bacillus stearothermophilusJo10-553. BseSI recognizes a degenerate hexanucleotide sequence 5[prime]-G(G/T)GC(A/C)[darr]C-3[prime] and cleaves DNA to produce 3[prime]-protruding tetranucleotide ends.
INTRODUCTION
Among the 221 different type II restriction endonucleases (1), there is a group of enzymes which cleave palindromic hexanucleotide sequences with ambiguous nucleotides at certain positions. At some positions of their recognition sequences these enzymes can be specified by purine:pyrimidine (R:Y) residues, such as 5[prime]-R[darr]CCGGY-3[prime] by Cfr10I (2), A/T:A/T (W:W) by StyI (5[prime]-C[darr]CWWGG-3[prime]) (3), A/C:G/T (M:K) by NspBII(5[prime]-CMG[darr]CKG-3[prime]) (4) or more complex sequences such as A/G/T:A/C/T (D:H) by SduI(5[prime]-GDGCH[darr]C-3[prime]) (5). Consequently, these enzymes cleave not one but several related nucleotide sequences. We describe here the isolation and characterization of a new representative of this group of enzymes, the thermophilic restriction endonuclease BseSIfrom Bacillus stearothermophilus, which recognizes a degenerate hexanucleotide 5[prime]-G(G/T)GC(A/C)[darr]C-3[prime] sequence and cleaves it to generate 4 nt protruding 3[prime]-ends.
METHODS AND RESULTS
Restriction endonuclease BseSI from B.stearothermophilus Jo10-553 has been purified using phosphocellulose, aminohexyl-Sepharose and heparin-Sepharose chromatography. To determine the substrate specifity of the enzyme, DNA substrates ([lambda] phage, pBR322, [phis]X174, M13mp18 and pUC57) were incubated at 55°C in 50 µl of MBI Fermentas G+ buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 0.1 mg/ml BSA) containing 1 µg DNA (Fig. 1A). The three BseSI cleavage sites on pBR322 DNA were mapped by double digestion with HindIII, BamHI, Eco88I, NdeI, Eam1105I and PstI (data not shown). The mapped positions (2289, 2787 and 4033) matched those cleaved by Alw44I, which recognizes the sequence 5[prime]-GTGCAC-3[prime]. BseSI also cleaved [phis]X174 DNA at the same position (4779) as Alw44I, but the cleavage patterns of [lambda] phage, M13mp18 and pUC57 DNAs were different. BseSI cleaved these substrates more frequently, indicating that its recognition sequence might be a degenerate sequence related to the Alw44I cleavage specificity, 5[prime]-GTGCAC-3[prime]. The single BseSI cleavage site in M13mp18 DNA, mapped to position 2090 using Eco47III and Mva1269I, was found to be 5[prime]-GGGCAC-3[prime]. In addition, pUC57 DNA was cleaved by BseSI at three Alw44I sites and one aditional sequence 5[prime]-GGGCCC-3[prime] at position 442. Based on the above mapping analysis experiments, we considered that the recognition sequence for BseSI was likely to be 5[prime]-G(G/T)GC(A/C)C-3[prime] (GKGCMC). The number and sizes of DNA fragments generated by BseSI digestion of phage [lambda] (11 fragments), pBR322 (three fragments), [phis]X174 (one fragment), M13mp18 (one fragment) and pUC57 (four fragments) were consistent with this sequence being the BseSI recognition sequence. Additional evidence supporting this conclusion was obtained after digestion of [lambda] DNA with BseSI, Alw44I (5[prime]-GTGCAC-3[prime]) and BmgI (5[prime]-GKGCCC-3[prime]). Alw44I and BmgI together cleave all BseSI sites. Triple digestion of any DNA substrate using these three enzymes should therefore yield the BseSI fragmentation pattern. Data presented in Figure 1B show that this was the case.
A
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B
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Figure 1. (A) Cleavage of various DNAs with BseSI. Lane 1, [lambda] DNA Eco130I digest as size markers; lane 2, [lambda] DNA + BseSI; lane 3, pBR322 DNA + BseSI; lane 4, [phis]X174 + BseSI; lane 5, M13mp18 DNA + BseSI; lane 6, pBR322 DNA Alw44I/MvaI digest as size markers; lane 7, pUC57 DNA + BseSI. (B) Triple digestion of [lambda] DNA with BseSI, Alw44I and BmgI. Lane 1, [lambda] DNA; lane 2, [lambda] DNA + BseSI; lane 3, [lambda] DNA + BseSI + Alw44I +BmgI; lane 4, [lambda] DNA + Alw44I + BmgI.
PBR322 DNA was used as a template to characterize the cleavage site of BseSI. A 20mer oligodeoxyribonucleotide complementary to pBR322 between positions 2380 and 2360 (ccw strand) was used in reverse sequencing through the BseSI site located at position 2289. Four dideoxy sequencing reactions (lanes G, A, T and C, respectively) using [[alpha]-33P]dATP were carried out (6). The same primer and template were used in a fifth non-terminating reaction, which also included T7 DNA polymerase, dNTP and [[alpha]-33P]dATP. The extension reaction was heat inactivated and then radiolabeled DNA was digested by BseSI and the reaction mix was divided into two. One sample was treated with T4 DNA polymerase. Both samples were diluted with sequencing dye and loaded on a standard sequencing gel together with the dideoxy sequencing reactions. The cleavage site of BseSI was determined by comparison of dideoxy sequencing ladders with fragments generated by BseSI cleavage and T4 DNA polymerase action on the digestion product.
Results of the determination of the BseSI cleavage site are shown in Figure 2. The fragment generated by BseSI digestion co-migrates with the A band of the sequence ladder (Fig. 2, lane 1) through the BseSI recognition site. From these results it can be inferred that BseSI cleaves DNA between the fifth and sixth nucleotides, starting from the 5[prime]-end of the BseSI recognition sequence, as indicated, 5[prime]-GKGCM[darr]C-3[prime]. Lane 2 shows the result when the fragment produced by BseSI digestion was further treated with T4 DNA polymerase. The single band obtained by the exonuclease activity of the T4 DNA polymerase co-migrates with the G band of the sequence ladder GTGCAC and indicates the cleavage point on the complementary DNA strand. These results would therefore indicate that DNA cleavage by BseSI generates 4 nt protruding 3[prime]-ends and that the cleavage specificity of BseSI is 5[prime]-GKGCM[darr]C-3[prime].
Figure 2. Determination of BseSI cleavage site. Lanes G, A, T and C, the sequence ladders through the BseSI site; lane 1, the product of the primed synthesis reaction cleaved with BseSI; lane 2, T4 DNA polymerase action on the BseSI digest.
DISCUSSION
5[prime]-GKGCM[darr]C-3[prime]is a new addition to the list of nucleotide sequences known to be recognized by restriction enzymes. BseSI should cleave DNA at all the Alw44I (5[prime]-GTGCAC-3[prime]), ApaI (5[prime]-GGGCCC-3[prime]) and BmgI (5[prime]-GKGCCC-3[prime]) sites.
ACKNOWLEDGEMENTS
The authors would like to thank Dr Michael Nelson for help in preparation of the manuscript. Special thanks are due to Ruta Jurgelyte for help in protein purification.
REFERENCES
*To whom correspondence should be addressed. Tel: +370 2 642 468; Fax: +370 2 642 624; Email: janulait{at}fermentas.lt
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