Figure 7.

Bifunctionality of the TspGWI: restriction and methylation activities of wt enzyme and selected mutants in the presence of divalent cations. Samples of 2 μg pBR322 DNA were incubated with TspGWI protein in the TspGWI reaction buffer (50 mM Tris-HCl 8.0, 10 mM DDT) supplemented with AdoMet, in the presence/absence of metal ions (Mg²⁺, Ca²⁺) or EDTA. Proteins were removed by proteinase K digestion, DNA was purified and every sample was divided into two equal parts. (A) Effect of Mg²⁺, Ca²⁺ on DNA restriction by TspGWI and its mutant variants. Lane M1, 1 kb DNA ladder (selected bands marked); lane 1 (-TspGWI; +EDTA), control 1 μg pBR322 DNA incubated in the reaction buffer devoid of Mg²⁺ and supplemented with 10 mM EDTA, no enzyme; lane 2 (-TspGWI; +Mg²⁺), Mg²⁺present, no enzyme; lane 3 (-TspGWI; +Ca²⁺), Ca²⁺present, no enzyme; lane 4 (+TspGWI; +EDTA), 2.5 μg (30 u) of native TspGWI, Mg²⁺ absent, supplemented with 10 mM EDTA; lane 5 (+TspGWI; +Mg²⁺), 2.5 μg of TspGWI, Mg²⁺ present; lane 6 (+TspGWI; +Ca²⁺), 2.5 μg of TspGWI, Ca²⁺ present; lane 7 (+D₅₈A; +EDTA), 2.5 μg TspGWI mutant D₅₈A, Mg²⁺ absent, supplemented with 10 mM EDTA; lane 8 (+D₅₈A; +Mg²⁺), 2.5 μg TspGWI mutant D₅₈A, Mg²⁺ present; lane 9 (+D₅₈A; +Ca²⁺), 2.5 μg TspGWI mutant D₅₈A, Ca²⁺ present; lane 10 (+N₄₇₃A; +EDTA), 2.5 μg TspGWI mutant N₄₇₃A, Mg²⁺ present, supplemented with 10 mM EDTA; lane 11 (+N₄₇₃A; +Mg²⁺), 2.5 μg TspGWI mutant N₄₇₃A, Mg²⁺ present; lane 12 (+N₄₇₃A; +Ca²⁺), 2.5 μg TspGWI mutant N₄₇₃A, Ca²⁺ present; lane M2, 100 bp DNA ladder (selected bands marked). (B) Effect of Mg²⁺, Ca²⁺ on DNA methylation by TspGWI and selected mutant variants. Lane M1, 1 kb DNA ladder (selected bands marked). Lanes 1–12: all samples were treated as described above in (A), DNA purified and subjected to digestion by 3 units of TspGWI for 1 h at 65°C in the TspGWI REase buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 10 mM DTT); lane M2, 100 bp DNA ladder (selected bands marked).