Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

Abstract

Background: We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.

Results: TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.

Conclusions: TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.

Figure 1

Limited proteolytic digestion of TspDTI. Purified native TspDTI was subjected to proteolytic digestion on immobilized TPCK-trypsin. Lane M, protein marker (GE Healthcare), bands marked: 97 kDa, phosphorylase b; 66 kDa, bovine serum albumin; 45 kDa, ovoalbumin; 31 kDa, carbonic anhydrase; 20.1 kDa, trypsin inhibitor; 14.4 kDa, lysozyme. Lane 1, proteolysis products. All the peptides obtained are marked with horizontal arrows. Of the 5 polypeptides obtained, only peptides 1, 2 and 3 were subjected to N-terminal sequencing.

Figure 2

SDS/PAGE analysis of the induction pattern of recombinant TspDTI endonuclease. Lane 1, control culture - crude lysate from E. coli expressing the cloned tspDTIRM gene, without induction (OD₆₀₀ = 0.7); lane 2, control culture after 12 h of cultivation; lane 3, crude lysate from E. coli expressing the cloned gene, before induction (OD₆₀₀ = 0.7); lane 4, crude lysate from E. coli expressing the cloned gene 3 h after induction; lane 5, crude lysate 6 h after induction; lane 6, crude lysate 12 h after induction; lane 7, purified, homogeneous recombinant TspDTI protein; lane M, protein marker (Thermo Fisher Scientific/Fermentas).

Figure 3

SDS/PAGE comparison of purified, recombinant homogeneous TspDTI, TsoI and TthHB27I (extended run on 6% gel). Lane M1, protein marker (GE Healthcare); lane M2, protein marker (Thermo Scientific); lane 1, TsoI; lane 2, TspDTI, lane 3, TthHB24I.

Figure 4

Sequence alignment between TspDTI and its close homologues in REBASE (BLAST E-value < 1E-40).

Figure 5

Schematic organization of the domains of the bifunctional TspDTI enzyme. (A) TspDTI domain architecture. (B, C, and D) Typical HsdR, HsdM, and HsdS subunit domain architecture, illustrated by way of example with EcoR124I.

Figure 6

Bifunctionality of TspDTI: restriction and methylation activities of the enzyme. (A) Effect of divalent metal cations on restriction activity of TspDTI. The DNA substrate used contains two TspDTI sites (→→). Partial digestion bands are marked in italics. Samples of 7.8 pmol 390 bp PCR fragment were incubated with an excess of TspDTI REase for 1 h at 70°C in 'primary TspDTI REase' buffer devoid of Mg²⁺ (10 mM Tris-HCl pH 8.0, 1 mM DTT) in the presence or absence of 50 μM SAM. The reaction buffer was supplemented with EDTA, Mg²⁺ or Ca²⁺ ions. Lane M2, 100 bp DNA ladder (selected bands marked); lane 1, undigested 390-bp PCR fragment; lane 2, incubation with TspDTI and Ca²⁺; lane 3, incubation with TspDTI, Ca²⁺ and SAM; lane 4, incubation with TspDTI and Mg²⁺; lane 5, incubation with TspDTI, Mg²⁺ and SAM; lane 6, incubation with TspDTI and EDTA; lane 7, incubation with TspDTI, EDTA and SAM. (B) MTase activity of TspDTI. The DNA substrate used contains two TspDTI sites (→→). Partial digestion bands are marked in italics. Samples of 7.8 pmol 390 bp PCR fragment were incubated with 78 pmol TspDTI protein in the TspDTI MTase buffer (10 mM Tris-HCl, pH 8.0, 1 mM DTT, 200 μM SAM) in the presence of either EDTA or Ca²⁺ ions. Proteins were removed by proteinase K digestion. The resulting DNA was purified and challenged with an excess of TspDTI REase for 1 h at 70°C in the 'primary TspDTI REase' buffer (10 mM Tris-HCl pH 8.0, 1 mM DTT) supplemented with 10 mM MgCl2; Lane M1, 1 kb DNA ladder (selected bands marked); lane M2, 100 bp DNA ladder (selected bands marked); lane 1, undigested 390-bp PCR fragment; lane 2, incubation of PCR fragment with TspDTI in REase buffer; lane 3, incubation with TspDTI in MTase buffer + EDTA/no subsequent incubation; lane 4, incubation with TspDTI in MTase buffer + Ca²⁺/no subsequent incubation; lane 5, incubation with TspDTI in MTase buffer + EDTA/subsequent incubation with TspDTI in REase buffer; lane 6, incubation with TspDTI in MTase buffer + Ca²⁺/subsequent incubation with TspDTI in REase buffer.

Figure 7

Effect of allosteric cofactors on TspDTI REase activity. 1 μg (= 5.58-pmol restriction sites) bacteriophage λ DNA was digested with 2.79 pmol TspDTI (0.5:1 M ratio of enzyme to recognition sites) for 30 min at 70°C and electrophoresed on 1.2% agarose/TBE gel. Lane M1, 1 kb ladder (selected bands marked); lane M2, 100 bp ladder (selected bands marked); lane K, undigested λ DNA; lane 1, (+ TspDTI, no allosteric cofactor; lane 2, (+ TspDTI, + 50 μM SIN); lane 3, (+ TspDTI, + 50 μM SAM); lane 4, (+ TspDT, + 50 μM SAH); lane 5, (+ TspDTI, + 50 μM ATP).