DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

Abstract

Topoisomerase II resolves intrinsic topological problems of doublestranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report singlemolecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg 2þ ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.he double helical nature of DNA imposes intrinsic topological problems during replication, repair, and transcription (1-3). Additionally, the topological state of the genetic material needs to be tightly regulated in order to promote proper biochemical interactions between DNA and a variety of proteins (1-4). Topoisomerases are enzymes that resolve topological problems within the double helix by repeated cycles of DNA cleavage and ligation (1-3, 5, 6).As a subclass of the topoisomerase family, type II topoisomerases are found in all organisms from bacteria to human, and even in some viruses (1-3, 5, 6). The essential roles of type II topoisomerases in cell metabolism, differences between bacterial and human homologues, and hyperactivation of these enzymes in cancer cells have been utilized for clinical treatments of bacterial infections and numerous cancers (3, 7-9).Extensive studies for more than twenty years have established that type II topoisomerases use a "two gate" mechanism for DNA strand passage (3, 10, 11), in which a DNA duplex (the transport or T-segment) is transported through an enzyme-mediated transient opening in a separate DNA duplex (the gate or G-segment). The directionality of strand passage is the N-terminal gate of the enzyme to the C-terminal gate. As a result of the double-stranded DNA passage mechanism, each catalytic event changes the linking number of DNA by two. The transport of the T-segment through the G-segment is thought to be initiated by the N-gate clamping motion induced by the binding of ATP to the enzyme (3,6,10,12).Although the double-stranded DNA breaks generated by type II topoisomerases are essential for the cellular functions of these enzymes, it is a dangerous process in which an aberrant operation can damage chromosomal integrity. In fact, widely prescribed anticancer and antibacterial drugs initiate cell death by increasing the cellular conce...

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

“…Although murine ES cells display TOP2A binding at promoters (Thakurela et al 2013), we do not find TOP2A CCR enrichment at promoters. Because TOP2A binding does not necessarily equate to cleavage (Mueller-Planitz and Herschlag 2007; Lee et al 2012), our study and the mouse ES cell study together suggest that TOP2A begins to track supercoil accumulation starting at the promoter but then cleaves well into the gene body during transcription elongation.…”

“…A key challenge to surmount is removal of the covalently attached DNA from TOP2 at the cleavage site. Conventional ChIP cannot distinguish noncovalent TOP2 DNA binding from TOP2 covalently bound to DNA in cleavage complexes, and most TOP2 DNA binding is noncovalent (Mueller-Planitz and Herschlag 2007;Lee et al 2012). General techniques to map DNA breaks in cells are not TOP2 specific, have low spatial resolution (Iacovoni et al 2010;Pang et al 2015), and may not detect TOP2 cleaved DNA ends protected by the enzyme (Crosetto et al 2013;Baranello et al 2014).…”

Iron Chelators with Topoisomerase-Inhibitory Activity and Their Anticancer Applications

“…Both type IA and type II topoisomerases require a divalent metal ion to function (51,227,239). For top2a, in vivo evidence has suggested that the DNA cleavage reaction requires Mg 2 + as a cofactor for DNA scission (143). The solved crystal structure of top2a also showed a single Mg 2 + atom per active site.…”

“…A separate metal ion is required for the interaction with ATP. The exact role played by the metal ions is still being explored and the importance of their interaction with other sites in the primase domain, for instance, remains unclear (143). Divalent ions Mn 2 + , Ca 2 + , and Co 2 + generated higher levels of DNA cleavage than Mg 2 + (49).…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…Two of the models, the Gsegment DNA-bending model (Vologodskii et al 2001) and the hooked-juxtaposition model (Buck and Zechiedrich 2004), propose that local geometry, such as bending by the topoisomerase or selection of Bhooked^conformations in juxtaposed DNA segments, results in preferential activity on linked DNA segments which, coupled with the ATPdependent unidirectional strand passage, results in global simplification of DNA topology. Structural studies and direct mechanical measurements have revealed that Topo IIA bend DNA upon binding (Dong and Berger 2007;Hardin et al 2011;Lee et al 2012Lee et al , 2013Thomson et al 2014) and thus that this enzyme likely has a higher binding affinity for bent DNA, such as at the apices of plectonemes in supercoiled DNA. To date, however, the proposed models cannot fully explain the below-equilibrium simplification phenomenon, indicating that the models may only partially reflect the mechanistic basis of this intriguing effect (Hardin et al 2011;Liu et al 2010;Martínez-García et al 2014;Seol et al 2013b;Thomson et al 2014;Yan et al 1999).…”