2.7. Top1 cleavage assay

An in vitro Top1 cleavage assay was performed as previously described [16]. A 45-nucleotide fragment containing the (TC)₃ sequence (5′-TTTGAAATACCGTGGCATCTCTCGTGACGAGTTACCATTTAAAGC) was labeled at the 3′ end prior to annealing to a complementary fragment (5′-GCTTTAAATGGTAACTCGTCACGAGAGATGCCACGGTATTTCAAA) at 1:1 ratio to produce the intact duplex substrate. The marker fragment (5′-GACGAGTTACCATTTAAAGC) was also 3′-end labeled. Human TOP1 (140 nM) was added to a reaction mixture containing 20–500 nM end-labeled DNA substrate, 10 mM Tris-HCl, 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 15 μg/ml BSA and 10% DMSO. 10 μM CPT was added to the reaction as indicated. Following incubation at 25°C for 1 hour, reactions were terminated by addition of 0.5% SDS. Samples were analyzed on a 20% polyacrylamide gel and labeled fragments were detected using a PhosphoImager.