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. Author manuscript; available in PMC: 2014 Feb 5.
Published in final edited form as: Biochemistry. 2013 Jan 23;52(5):984–991. doi: 10.1021/bi301643h

Figure 8. Aminoalkylation of Cys167 restores supercoil relaxation activity.

Figure 8

Relaxation reaction mixtures containing (per 20 μl) 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 300 ng of supercoiled pUC19 DNA, and 2.25 ng of WT, K167C, 2-BEA-treated K167C or 3-BPA-treated K167C TopIB as specified were incubated at 37°C. The reactions were initiated by the addition of enzyme. Aliquots (20 μl) were withdrawn at the times specified and quenched immediately with SDS. The time 0 samples were taken prior to addition of enzyme. The reaction products were analyzed by 1% agarose horizontal gel electrophoresis. The gels were stained with ethidium bromide and DNA was visualized under UV illumination.