A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

Abstract

Type IB topoisomerases (Top1Bs) relax excessive DNA supercoiling associated with replication and transcription by catalyzing a transient nick in one strand to permit controlled rotation of the DNA about the intact strand. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Top1B (nTop1). Previous work on vaccinia Top1B led to an elegant model that describes a straightforward dependence of rotation and religation on the torque caused by supercoiling. Here, we used a singlemolecule DNA supercoil relaxation assay to measure the torque dependence of nTop1 and its inhibition by CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTop1. Despite substantial sequence homology in their core domains, nTop1 and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and CPT, with the N-terminal deletion mutant of nTop1 showing intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torqueindependent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation and the efficiency of CPT binding. This "kinetic clutch" mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.cancer therapy | magnetic tweezers | single-molecule biophysics T ype IB topoisomerase (Top1B) is essential for relaxing DNA supercoils generated during replication, transcription, DNA recombination, and repair (1-4). Due to its prominent roles in DNA metabolism, human Top1B is an important target for cancer therapies (2, 4, 5). Top1B relaxes DNA supercoils by forming a clamp around the DNA and generating a transient nick, which allows rotation of the DNA until the nick is religated (6, 7). This DNA rotation was thought to be hindered by interactions with the protein clamp because the cross-section of rotating DNA at the nick increases twofold during rotation (6,8,9). This was confirmed in a pioneering single-molecule study of relaxation by vaccinia Top1B that led to a model of DNA rotation as a random walk along a rugged energy landscape associated with the rotation angle of the 5′ end of the DNA. In this model, the rotation rate is dictated by the escape from multiple energy wells; religation is in kinetic competition with rotation at a specific energy well. Torque on the DNA (i.e., supercoiling) tilts the energy landscape, lowering the rotation energy barriers, thereby increasing the rotation rate and decreasing the probability of religation. As a result, both the rotation rate and the religation probability are torque-dependent to different degrees. This simple yet elegant model describes vaccinia Top1B supercoil relaxation as a fu...

The dynamic interplay between DNA topoisomerases and DNA topology

“…Topo V (topoisomerase Type IC) shares a similar catalytic mechanism with Topo IB despite the lack of structural or sequence similarity (Schoeffler and Berger 2008). DNA torsion lowers the rotational energy barrier (Wereszczynski and Andricioaei 2010) and thus enhances the rate of rotation, i.e., the relaxation rate for both types of topoisomerases (Koster et al 2005;Seol et al 2012;Taneja et al 2007). DNA twist also affects the efficacy of human Topo IB inhibitors, some of which are Federal Drug Administration-approved chemotherapeutic agents that act by preventing religation of the cleaved DNA and trapping the cleavage complex comprised of the topoisomerase, DNA, and inhibitor (Pommier and Cushman 2009).…”

“…In addition, geometric differences between positive and negative Wr or DNA juxtapositions within knotted or catenated DNA are subject to chiral-and geometric-dependent activities of some Type II topoisomerases. For example, topoisomerase IV has been shown to be sensitive to the DNA crossing angle α depicted in the figure inhibitor-mediated cleavage with supercoiled DNA may reflect the torque-dependent increase in the occupancy of a cleaved, but not rotating, DNA-protein conformation that promotes inhibitor binding (Seol et al 2012). Supercoilingmediated effects have also been demonstrated for the inhibition of human Topo IIα and Topo IIβ, the two isoforms of human Topo IIA, by inhibitors (e.g., etoposide) that act to prevent religation and stabilize enzyme-mediated cleavage complexes in a similar manner as the Topo IB inhibitors (McClendon and Osheroff 2006).…”

Biochemical Characterization of the Topoisomerase Domain of Methanopyrus kandleri Topoisomerase V

“…Type IB enzymes form a phosphotyrosyl bond with the 3Ј end of the broken DNA strand, relax either positively or negatively supercoiled DNA, and do not require divalent metal cations for activity (6). Type IB topoisomerases remove supercoils by a swiveling or "controlled rotation" mechanism (21)(22)(23)(24), allowing the cleaved ssDNA to rotate around the uncleaved single strand and thus changing the linking number by several steps during each reaction cycle. The active site of all type IB topoisomerases contains a highly conserved pentad of residues, consisting of a Tyr, two Arg residues, a Lys, and either a His in human and viral proteins or an Asn in bacterial homologs.…”

F10 Cytotoxicity Via Topoisomerase I Cleavage Complex Repair Consistent With a Unique Mechanism for Thymineless Death

“…Elucidating the DNA repair pathways activated in cells treated with F10 relative to CPT can provide insight into how Top1 poisoning induced by fluoropyrimidine drugs differs from traditional Top1 poisons, and also clarify which aspects of the DNA damage response are activated by Top1-independent processes. Top1 relieves DNA torsional stress generated during replication and transcription by nicking supercoiled DNA allowing relaxation to proceed via controlled rotation [3], and then re-ligating the nick [4] to restore the DNA duplex. CPT forms a ternary complex with Top1 and the nicked DNA resulting in formation of a Top1 cleavage complex (Top1cc) that, if not repaired, can lead to DNA double-strand breaks (DSBs) through collision with advancing…”

“…Top1 relieves DNA torsional stress generated during replication and transcription by nicking supercoiled DNA allowing relaxation to proceed via controlled rotation [3], and then re-ligating the nick [4] COmmEntaRY Gmeiner, Gearhart, Pommier & Nakamura future science group replication forks [5]. Top1cc also form upon F10 treatment as Top1 acts upon DNA in which FdU has become incorporated proximal to Top1 cleavage sites [6].…”