Topoisomerase I

Topoisomerase I (DNA-relaxing enzyme) catalyzes the removal of superhelical turns from covalently closed DNA by a transient breakage and rejoining of phosphodiester bonds. Topoisomerase I is active in the presence of EDTA.

Applications: Relaxing positively and negatively supercoiled DNA (1). Producing DNA topoisomers (2).

Source: Purified from calf thymus.

Performance and Quality Testing: Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease, and phosphatase assays; conversion of super-coiled DNA to relaxed DNA.

Unit Definition: One unit catalyzes the conversion of 0.5 μg of superhelical Φ X174 RF DNA to a relaxed state in 30 min. at 37°C.

Unit Reaction Conditions: 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl₂ , 0.1 mM EDTA, 0.5 mM DTT, 30 μg/ml/BSA, 0.5 μg
Φ X174 RF DNA, and enzyme in 50 μl for 30 min. at 37°C.