Chiral discrimination and writhe-dependent relaxation mechanism of human topoisomerase IIα.

BACKGROUND: Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils. RESULTS: Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity. CONCLUSION: Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains. SIGNIFICANCE: Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase IIα. Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and "braided" DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases.

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I.

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as 'topological poisons of topoisomerase I'.

Metnase promotes restart and repair of stalled and collapsed replication forks.

Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.

Interactions between the etoposide derivative F14512 and human type II topoisomerases: implications for the C4 spermine moiety in promoting enzyme-mediated DNA cleavage.

F14512 is a novel etoposide derivative that contains a spermine in place of the C4 glycosidic moiety. The drug was designed to exploit the polyamine transport system that is upregulated in some cancers. However, a preliminary study suggests that it is also a more efficacious topoisomerase II poison than etoposide [Barret et al. (2008) Cancer Res. 68, 9845-9853]. Therefore, we undertook a more complete study of the actions of F14512 against human type II topoisomerases. As determined by saturation transfer difference (1)H NMR spectroscopy, contacts between F14512 and human topoisomerase IIα in the binary enzyme-drug complex are similar to those of etoposide. Although the spermine of F14512 does not interact with the enzyme, it converts the drug to a DNA binder [Barret et al. (2008)]. Consequently, the influence of the C4 spermine on drug activity was assessed. F14512 is a highly active topoisomerase II poison and stimulates DNA cleavage mediated by human topoisomerase IIα or topoisomerase IIß. The drug is more potent and efficacious than etoposide or TOP-53, an etoposide derivative that contains a C4 aminoalkyl group that strengthens drug-enzyme binding. Unlike the other drugs, F14512 maintains robust activity in the absence of ATP. The enhanced activity of F14512 correlates with a tighter binding and an increased stability of the ternary topoisomerase II-drug-DNA complex. The spermine-drug core linkage is critical for these attributes. These findings demonstrate the utility of a C4 DNA binding group and provide a rational basis for the development of novel and more active etoposide-based topoisomerase II poisons.

Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal.

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1-206 or 191-206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621-6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation.

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.