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Top1-Clamp is sensitive to CPT. Top1, Top1-Clamp, and Top1Y723F were incubated in DNA-cleavage reaction mixtures with a single 3′ 32P-labeled DNA fragment (18). As indicated, reaction mixtures contained 50 μM CPT, 5 mM diamide, or 100 mM DTT. After 10 min at 37°C, covalent-complex formation was assessed in sequencing gels by PhosphorImager analysis. The asterisk indicates a high-affinity cleavage site. Arrows indicate novel cleavage sites.
