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.

Human topoisomerase IIα, but not topoisomerase IIβ, 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 IIα and β 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 IIα CTD in the recognition of DNA topology, hTop2αΔ1175, 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 IIβ was replaced with that of the α isoform, the resulting enzyme preferentially relaxed positively supercoiled substrates. In contrast, a chimeric topoisomerase IIα that carried the CTD of the β isoform lost its ability to recognize the geometry of DNA supercoils during relaxation. These findings demonstrate that human topoisomerase IIα 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 IIα 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.Topoisomerases are essential enzymes that modulate the topological state of DNA in the cell (1-7). Type II enzymes act by passing an intact double helix through a transient double-stranded break that they generate in a separate segment of DNA (3,4,(7)(8)(9). As a consequence of their double-stranded DNA passage reactions, type II topoisomerases are able to alleviate torsional stress in duplex DNA and remove knots and tangles from the genetic material (1-7).Based on amino acid comparisons to prokaryotic type II enzymes, eukaryotic topoisomerase II can be divided into three domains (1,4,7,(10)(11)(12). The N-terminal (or ATPase) domain

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 [1H]-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 IIβ. 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.

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