Topoisomerase I and II Activity Assays

DNA topoisomerases I and II (top1 and top2, respectively) are ubiquitous enzymes that play an essential role in transcription, replication, chromosome segregation, and DNA repair. The basic enzymatic reaction of topoisomerases, namely reversible DNA nicking, is a transesterification reaction where a DNA phosphodiester bond is transferred to a specific enzyme tyrosine residue. Eukaryotic top1 and top2 exhibit major differences concerning their mechanism of action. Top1 acts as a monomer and forms a covalent bond with the 3′-terminus of a DNA single-strand break ( 1 – 3 ) whereas top2 acts as an homodimer and forms a covalent bond with the 5′-terminus of the DNA double-strand break with a four base-pairs overhang (Fig. 1 ) ( 1 – 4 ). No energy cofactor is required for top1 activity, whereas top2 hydrolyzes adenosine triphosphate (ATP) during its catalytic cycle.   Fig. 1.  Top1- and top2-cleavage complexes. ( A ) Top1 acts as a monomer, makes a single-strand break and covalently binds to the 3′-end of the break, leaving a 5′-hydroxyl end. ( B ) Top2 acts as a dimer, and generally makes a double-strand break. Each strand is cleaved by one monomer, with a 4-base overhang. Each monomer covalently binds to the 5′-end of the break and leaves a 3′-hydroxyl end.