The QTK loop is essential for the communication between the N-terminal atpase domain and the central cleavage--ligation region in human topoisomerase IIalpha.

We have characterized a human topoisomerase IIalpha enzyme with a deletion of the conserved QTK loop, which extends from the transducer domain to the ATP-binding pocket in the GHKL domain. The loop has been suggested to play a role for interdomain communication in type II topoisomerases. The mutant enzyme performs only very low levels of strand passage, although it is able to cleave and ligate DNA as well as close the N-terminal clamp. Cleavage is nearly unaffected by ATP and ATP analogues relative to the wild-type enzyme. Although the enzyme is able to close the clamp, the clamp has altered characteristics, allowing trapping of DNA also in the absence of an ATP analogue. The enzyme furthermore retains intrinsic levels of ATPase activity, but the activity is not stimulated by DNA. Our observations demonstrate that the QTK loop is an important player for the interdomain communication in human topoisomerase IIalpha. First, the loop seems to play a role in keeping the N-terminal clamp in an open conformation when no nucleotide is present. Once the nucleotide binds, it facilitates clamp closure, although it is not essential for this event. The QTK loop, in contrast, is essential for the DNA-stimulated ATPase activity of human topoisomerase IIalpha.

Bimodal recognition of DNA geometry by human topoisomerase II alpha: preferential relaxation of positively supercoiled DNA requires elements in the C-terminal domain.

Human topoisomerase IIalpha, but not topoisomerase IIbeta, can sense the geometry of DNA during relaxation and removes positive supercoils >10-fold faster than it does negative superhelical twists. In contrast, both isoforms maintain lower levels of DNA cleavage intermediates with positively supercoiled substrates. Since topoisomerase IIalpha and IIbeta differ primarily in their C-terminal domains (CTD), this portion of the protein may play a role in sensing DNA geometry. Therefore, to more fully assess the importance of the topoisomerase IIalpha CTD in the recognition of DNA topology, hTop2alphaDelta1175, a mutant human enzyme that lacks its CTD, was examined. The mutant enzyme relaxed negative and positive supercoils at similar rates but still maintained lower levels of cleavage complexes with positively supercoiled DNA. Furthermore, when the CTD of topoisomerase IIbeta was replaced with that of the alpha isoform, the resulting enzyme preferentially relaxed positively supercoiled substrates. In contrast, a chimeric topoisomerase IIalpha that carried the CTD of the beta isoform lost its ability to recognize the geometry of DNA supercoils during relaxation. These findings demonstrate that human topoisomerase IIalpha recognizes DNA geometry in a bimodal fashion, with the ability to preferentially relax positive DNA supercoils residing in the CTD. Finally, results with a series of human topoisomerase IIalpha mutants suggest that clusters of positively charged amino acid residues in the CTD are required for the enzyme to distinguish supercoil geometry during DNA relaxation and that deletion of even the most C-terminal cluster abrogates this recognition.

Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level.

We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.