Inhibition of Topoisomerase I Cleavage Activity by Thiol-reactive Compounds

Abstract

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.Eukaryotic DNA topoisomerase I (Top1) 3 is an essential enzyme involved in the regulation of DNA topology associated with most DNA transactions, including replication, transcription, recombination, and chromatin remodeling (1-3). This role is dependent upon the ability of Top1 to introduce transient single strand breaks in duplex DNA via the formation of a covalent bond between the 3Ј-phosphate of the cleaved strand and a tyrosine residue of the enzyme (Tyr-723 in human) (4). Within the covalent Top1-DNA complex, rotation of the broken strand around the uncleaved strand results in DNA supercoil relaxation (5, 6). DNA continuity is then restored by Top1-catalyzed religation of the 5Ј-hydroxyl termini. Under normal conditions, the DNA cleavage step of the DNA cleavage/religation equilibrium is rate-limiting, which results in the detection of a small fraction of cleavage complexes at any given time (7). Top1 poisons, such as camptothecin (CPT) and its analogs, reversibly stabilize the Top1-DNA complex by inhibiting DNA religation (8, 9). Collision of the stabilized complexes with advancing replication forks leads to the formation of irreversible strand breaks that are ultimately responsible for cell death (10, 11). Conversely, catalytic inhibitors exert their cytotoxicity by preventing Top1 binding to the DNA and/or Top1 cleavage, resulting in the inhibition of DNA relaxation. This is the case for DNA binders and DNA intercalators,...

Oxidation Status of Human OGG1-S326C Polymorphic Variant Determines Cellular DNA Repair Capacity

“…To explore whether other cysteines within OGG1 can potentially interact with Cys326 to form a disulfide bond, cell extracts were treated with phenylarsine oxide (PAO), a reagent that specifically cross-links cysteines located in close proximity by forming stable dithioarsine ring bridge (30). Having a cysteine in position 326 rendered the OGG1 DNA glycosylase activity of lymphoblastoid cells more sensitive to PAO (Fig.…”

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

“…On the other hand, catalytic inhibitors act through inhibiting any other step of the Top1 enzymatic cycle. They include both natural and non-natural compounds [7] and they mainly act by inhibiting the cleavage [8–10] or the DNA binding [11,12], although some of them are able to inhibit both cleavage and religation [13,14]. The CPT derivatives are widely used in clinics; however, they have some intrinsic limits, the main one being linked to the equilibrium of the α-hydroxylactone E-ring of CPTs with the carboxylate form, which is inactive against Top1 and tightly binds to serum albumin.…”

Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin

“…For example, as depicted in Fig. 6A, Top1p cleavage of a high affinity site within a suicide DNA substrate liberates a dinucleotide and traps the covalent Top1p-DNA complexes (6,18,38). In this case, Top1-DNA covalent complex formation is monitored by the accumulation of a peptide-linked 14-mer, produced by trypsin digestion of the reaction products in the presence of urea (39).…”