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.