A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI, TaqII and Tth111II

Abstract

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-3', was isolated from Thermus sp. DT. TspDTI appears to be a 'twin' of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5'-ACGGA(N(11/9))-3' and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9-100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5-122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.

Figure 1

Partial digestion of pUC19 plasmid DNA with TspDTI restriction endonuclease. (A) TspDTI cleavage of pUC19 DNA, 1.5% agarose/TAE. Lane 1, 1 kb ladder; lane 2, 100 bp ladder; lane 3, untreated pUC19 DNA; lane 4, TspDTI-cut pUC19 DNA. (B) TspDTI cleavage of pUC19 DNA, 6% polyacrylamide/TBE. Lane 1, 100 bp ladder; lane 2, untreated pUC19 DNA; lane 3, TspDTI-cut pUC19 DNA. The smallest partial digestion band of 376 bp is indicated in bold italics with horizontal arrow.

Figure 2

Determination of polypeptide molecular sizes for Thermus class-IIS endonucleases: TspDTI, TspGWI, TaqII and Tth111II. (A) SDS/PAGE of purified, homogeneous TspDTI, TspGWI, TaqII and Tth111II endonucleases. Lane M1, protein marker broad range (New England Biolabs); lane M2, low molecular weight marker (Amersham-Pharmacia). Bands marked in lanes M1 and M2 are as follows: 158.2 kDa, MBP-β-galactosidase; 116.4 kDa, β-galactosidase; 97.2 kDa, phosphorylase b; 66.4 kDa, bovine serum albumin. Lane 1, TspGWI endonuclease; lane 2, TspDTI endonuclease; lane 3, Tth111II endonuclease; lane 4, TaqII endonuclease. (B) Graph showing estimation of polypeptide sizes for TspDTI, TspGWI, TaqII and Tth111II.

Figure 3

Amino acid sequence comparison between two tryptic fragments of TspGWI endonuclease and complete TaqII endonuclease sequence. Identical amino acid residues are indicated by straight lines, and similar residues by dots. The partial amino acid sequence of TspGWI restriction endonuclease was obtained by limited trypsin digestion, followed by N-terminal protein sequencing of two internal peptides. The TaqII amino acid sequence was translated from the cloned taqIIR coding gene (; S.M.Rutkowska, I.Jaworowska, I.Sobolewski and P.M.Skowron, manuscript in preparation). The first homologous region extends from amino acid 30 to 37 and the second region from amino acid 445 to 453 of TaqII restriction endonuclease.