Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality

Abstract

Eukaryotic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable ''lip'' domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Top1-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369 -Glu-497 salt bridge. In reducing environments, Top1-Clamp was catalytically active. However, contrary to crosslinking the salt-bridge loops [Carey, J. D NA topoisomerases alter DNA topology by the concerted cleavage and religation of DNA strand(s) (reviewed in refs. 1-3). The nucleophilic attack of an active-site tyrosine on a phosphodiester DNA bond generates a phosphotyrosyl linkage, providing a transient protein-linked gate through which another DNA strand or duplex may pass. A second transesterification resolves the tyrosyl-DNA linkage to restore DNA integrity. DNA topoisomerases play critical roles in most DNA transactions, including DNA replication, transcription, and recombination, as well as chromosome segregation. These enzymes also constitute the targets of clinically important anticancer and antibacterial agents (4-6). With human DNA topoisomerase I (Top1), camptothecin (CPT) analogues (topotecan and CPT-11) have shown remarkable antitumor activity against pediatric and adult malignancies (7). These agents poison Top1 by reversibly stabilizing the covalent enzyme-DNA complex, inducing S-phase-dependent cell lethality.Type IB enzymes, including eukaryotic and vaccinia virus Top1, are distinct from other enzymes in that the active-site tyrosine becomes linked to a 3Ј-phosphoryl end, versus the 5Ј-phosphotyrosine bond formed by types IA and II enzymes (1-3). Crystallographic data indicate that the monomeric enzymes (types IA and IB) or the protein subunits that constitute the symmetrical halves of type II enzymes [DNA gyrase and DNA topoisomerase II (Top2)] form a protein clamp (1-3). Yet, type IB enzymes are again unique in that biochemical and structural data support a mechanism of DNA rotation, as opposed to the coordinated domain movements in types IA and II enzymes that are necessary for an enzyme-bridging mechanism (1, 3, 8).The conserved central domain of eukaryotic Top1 forms the protein clamp (Fig. 1). Subdomains I and II constitute the upper cap, which is connected by a flexible hinge to the bottom part of the clamp, subdomain III. Two extended ␣-helices connect the clamp core with the conserved C-terminal domain, containing the activesite tyrosine. Human Top1 struc...

Substitution of Conserved Residues within the Active Site Alters the Cleavage Religation Equilibrium of DNA Topoisomerase I

“…Thus, some flexibility of Top1p protein domains is required for enzyme binding of DNA and the rotation of DNA strands necessary to effect changes in DNA topology. These studies further demonstrated that expression of the catalytically inactive Top1Y723Fp-clamp in yeast cells with elevated levels of oxidized glutathione, also sufficed to induce cell lethality independent of covalent complex formation (22).…”

“…Using molecular modeling to design a reversible disulfide bond across the opposable lip domains that complete the clamp, we recently demonstrated that DNA rotation is inhibited within the locked Top1p⅐clamp⅐DNA complex (22). Thus, some flexibility of Top1p protein domains is required for enzyme binding of DNA and the rotation of DNA strands necessary to effect changes in DNA topology.…”

Repair of Topoisomerase I‐Mediated DNA Damage

“…Crystal structures of Top1 [20][21][22] show the enzyme encircling the DNA tightly like a clamp (Fig. 3D), which accounts for the fact that Top1 controls the processive relaxation of supercoiled DNA [20,[23][24][25] Two key pharmacological properties of camptothecins need to be stressed. First, camptothecins bind reversibly to the Top1 cleavage complexes.…”

Inhibition of Topoisomerase I Cleavage Activity by Thiol-reactive Compounds

“…Moreover, considerable structural flexibility is necessary for enzyme binding of duplex DNA and rotation of the cleaved DNA within the Top1 protein. This important notion derives from crystal structures of human Topo70 in complex with DNA (49,50) and studies based on the reversible cross-linking of the human Top1 protein clamp (28). Thus, it is possible that distant cysteinyl residues of the primary structure of the enzyme might come closer together and become a target for PAO.…”

“…Top1 Protein Purification-Full-length human Top1, Top1C504A, Top1C505A, Top1C504A,C505A, and Top1Y723F proteins, all containing an N-terminal FLAG epitope, were partially purified as described (28) from galactose-induced cultures of top1⌬ yeast cells, transformed with YCpGAL1-eTOP1 vectors. To obtain homogeneous protein preparations, Top1 fractions were applied to an anti-FLAG M2 affinity gel (Sigma), and the proteins were eluted with an excess of FLAG peptide in TBS (50 mM Tris, pH 7.4, 150 mM KCl).…”