Assay of cleavage of DNA substrate by EcTopA

The G-strand or T-strand oligonucleotide of Substrate-Y was labeled with ³²P at the 5′-end with γ-³²P-ATP and T4 polynucleotide kinase before hybridization with the unlabeled opposite strand. Substrate-Y formed by hybridization was then incubated with EcTopA in 10 μl of 20 mM sodium phosphate, pH 7.2, 1 mM EDTA with 10 or 100 mM NaCl at room temperature for 30 min. The reactions were stopped by addition of an equal volume of sequencing gel loading buffer and electrophoresed in a 15% denaturing sequencing gel, followed by Phosphorimager analysis.