The mechanism of topoisomerase I poisoning by a camptothecin analog

Abstract

We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (؊1) and downstream (؉1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme-substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure-activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site. Eukaryotic DNA topoisomerase I (topo I) is an enzyme that acts to relax supercoils generated during transcription and DNA replication (1). Because of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Topo I mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723) at a DNA phosphodiester bond forming a 3Ј-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5Ј-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3Ј-phosphotyrosyl topo I-DNA complex remains low (2).However, a variety of DNA lesions and drugs have been shown to stabilize the covalent 3Ј-phosphotyrosyl intermediate (3). For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity (4) and was later demonstrated to cause the accumulation of topo I-DNA adducts in vitro and in vivo (5, 6). CPTs bind the covalent 3Ј-phosphotyrosyl intermediate and specifically block DNA religation (7), thus converting topo I into a DNA-damaging agent (8). Topo I is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific (9). During DNA replication, the replication fork is thought to collide with the ''trapped'' topo I-DNA complexes, resulting in double-strand breaks and ultimately cell death (10).It has been difficult to study the mechanism of CPT activity because the drug acts as an uncompetitive inhibitor and binds only the transient covalent enzyme-substrate complex (7,11). To isolate the covalent topo I-DNA complex, we have used suicide DNA ...

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

“…Mutation of residues, such as Gly-363, within the Top1 upper lip domain has been reported to alter enzyme sensitivity to CPT, producing a drug-resistant phenotype (12,16,23,24). The structure of Top1-DNA-topotecan indicates that the 365-loop stabilizes an intercalation pocket within Top1 to allow the rotation of DNA caused by drug binding (11). These results predict that the G365C mutation would reduce enzyme sensitivity to CPT, which was borne out in DNA cleavage assays (data not shown) and in top1⌬ yeast expressing Top1G365C (see Fig.…”

“…CPT targets Top1 by reversibly stabilizing the covalent enzyme-DNA intermediate (1,5,6,11). Mutation of residues, such as Gly-363, within the Top1 upper lip domain has been reported to alter enzyme sensitivity to CPT, producing a drug-resistant phenotype (12,16,23,24).…”

“…Two extended ␣-helices connect the clamp core with the conserved C-terminal domain, containing the activesite tyrosine. Human Top1 structures are cocrystals of a C-terminal 70-kDa fragment of the enzyme (Topo 70, a 70-kDa form of human Top1 that is missing the nonessential N terminus), or reconstituted core and active-site tyrosine domains, with duplex DNA packaged tightly into a central pore 15-20 Å in diameter (8)(9)(10)(11). Opposable ''lip'' domains (one loop from subdomain I and two loops from subdomain III) interact with phosphates in the DNA and form a salt bridge to complete the circumnavigation of Top1 around the DNA.…”

An unusual type IB topoisomerase from African trypanosomes

“…Like other IBs, the T. brucei topoisomerase has a Lys at 468L, not the His seen in recombinases (26). Also conserved are some (Gly 252L , Asp 416L , Asn 232S ), but not all (e.g., Asp 535L and Val 239S ), of the residues important for camptothecin sensitivity (27). Perhaps the latter contribute to the observed differences in susceptibility to camptothecin analogs (14), which originally prompted these studies.…”

Determination of the Protein-Binding Properties of Camptothecins by Means of Optical Spectroscopy Methods

“…CPT was isolated in the 1960s from a tree Camptotheca acuminate [2]. Interest in camptothecin increased in the 1980s and 90s, after the discovery of its mechanism of action based on inhibition of nuclear enzyme topoisomerase I which is involved in DNA replication [3]. This interest resulted in the synthesis of many analogues, two of which were introduced as a medicine in anticancer therapy (topotecan, irinotecan), while some other are at the stage of clinical trials [4].…”