Figure 8
Evaluation of the effect of cofactor SAM and its analogues on TsoI activity. 0.3 μg (= 0.6 pmol recognition sites) single site PCR substrate (390 bp) was digested with decreasing amounts of wt TsoI for 16 h at 55°C in the standard reaction buffer: 10 mM Tris–HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 0.01 mg/ml BSA, 0.5 mM DTTn the presence or absence of SAM. (A) DNA cleavage in the absence of SAM. Lane M1, GeneRuler™ 1 kb DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, undigested PCR fragment; lanes 1–7, digested PCR fragment: lane 1, with 4.8 pmol; lane 2, with 2.4 pmol; lane 3, with 1.2 pmol; lane 4, with 0.6 pmol; lane 5, with 0.3 pmol; lane 6, with 0.15 pmol; lane 7, with 0.075 pmol; lane M2, GeneRuler™ 100 bp Plus DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked). (B) DNA cleavage in the presence of 50 μM SAM. Lane M1, GeneRuler™ 1 kb DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, undigested PCR fragment; Lanes 1–7, samples were digested with decreasing amounts of TsoI as described in (A); lane M2, GeneRuler™ 100 bp Plus DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked). (C) The influence of enzyme to recognition site ratio on TsoI DNA cleavage in the presence or absence of SAM. (D) Effect of allosteric cofactors on wt TsoI REase activity. Three putative cofactors or analogues (SAM, SAH, SIN) as well as ATP were compared for their influence on TsoI DNA digestion activity. 0.5 μg of λ DNA (= 0.016 pmol recognition sites) was digested with 0.7 pmol (0.048 u) of TsoI in standard TsoI buffer supplemented with 50 μM of the appropriate effector and 0.5 mM DTT for 30 min at 55°C. Lane M, GeneRuler™ 1 kb DNA Ladder (Thermo Fisher Scientific (Fermentas); selected bands marked); lane K, untreated λ DNA; lane 1, λ DNA cleaved with wt TsoI (no cofactors, except Mg2+); lane 2, (+TsoI wt, +SAM); lane 3, (+TsoI wt, +SIN); lane 4, (+TsoI wt, +SAH); lane 5, (+TsoI wt, +ATP). DNA was treated with low amount of TsoI to pinpoint differences in the stimulatory effect. The reaction products were resolved on 1.2% agarose gel in TBE buffer and stained with EtBr.