Figure 7

Bifunctionality of TaqII: REase/MTase activities of the enzyme. (A) Evaluation of cofactor SAM effect and its analogues on TaqII activity. Three putative effectors and ATP, were compared in their effect on syn-TaqII REaseactivity. 300 ng of the PCR fragment (390 bp; = 1.2-pmol of 5′-GACCGA-3′ recognition sites) was digested with 17 pmol (0.12 u) of syn-TaqII as described in Methods. Lanes M, modified GeneRuler™ 100 bp DNA Ladder (Thermo Fisher Scientific/Fermentas); lane K, untreated DNA; lane 1, + syn-TaqII (no cofactors, except Mg²⁺); lane 2, as in lane 1 + SIN); lane 3, + SAM; lane 4, + SAH; lane 5, + ATP. DNA was treated with limited amounts of syn-TaqII, to pinpoint stimulatory effect differences. (B) The MTase activity of syn-TaqII. Samples of 1.2 pmol 390 bp PCR fragment were incubated with 30 pmol syn-TaqII protein in the MTase buffer in the presence of either EDTA or Ca²⁺ as described in Methods. The resulting DNA was purified and challenged with an excess of TaqII REase: 1.18 pmoles the enzyme and 0.6 pmoles 5′-GACCGA-3′ sites (2 : 1 molar ratio) for 1 h at 65°C in the optimal TaqII REase buffer supplemented with 10 mM MgCl₂; Lane M, as in panel A; lane K1, untreated DNA; lane K2, no TaqII/ REase buffer; lane 1, + TaqII, MTase buffer + EDTA/subsequent incubation + TaqII, REase buffer; lane 2, + TaqII, MTase buffer + EDTA/no subsequent incubation; lane 3, + TaqII, MTase buffer + EDTA/subsequent incubation with TaqII, REase buffer; lane 4, no TaqII, MTase buffer + Ca²⁺/subsequent incubation + TaqII, REase buffer; lane 5, + TaqII, MTase buffer + Ca²⁺/no subsequent incubation; lane 6, + TaqII, MTase buffer + Ca²⁺/subsequent incubation + TaqII, REase buffer.