Figure 2.

Analysis of biochemical properties of MtTOP1-G116 S enzyme and the lethal consequence of its overexpression. (A) Magnesium dependence of DNA cleavage. Wild-type and G116 S MtTOP1 proteins were assayed for cleavage of negatively supercoiled plasmid DNA with agarose gel electrophoresis in the presence of 0.5 μg/ml ethidium bromide. N: nicked DNA; S: supercoiled DNA (B) MtTOP1-G116 S mutant has no relaxation activity even in the presence of excess Mg(II) ions. Wild-type and G116 S mutant MtTOP1 (400 ng) were assayed in a 20 μl reaction with 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml gelatin and the indicated concentration of MgCl₂ at 37°C for 30 min. Agarose gel electrophoresis in the absence of ethidium bromide was used to separate the supercoiled DNA from the slower migrating relaxed topoisomers. (C) Loss of viability of E. coli JD5 after induction of MtTOP1-G116 S expression with arabinose. Viable colony counts determined 2 h after addition of 0.2% arabinose were divided by viable colony counts from control non-induced cultures to obtain the survival ratio.