Alterations in Linker Flexibility Suppress DNA Topoisomerase I Mutant-induced Cell Lethality

Losasso, Carmen; Cretaio, Erica; Palle, Komaraiah; Pattarello, Luca; Bjornsti, Mary-Ann; Benedetti, Piero

doi:10.1074/jbc.m608200200

Cited by 24 publications

(24 citation statements)

References 40 publications

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“…These data suggest a dynamic interplay between active site ␣-helical structure and linker flexibility attends drug binding to the covalent Topo70-DNA complex. Such long range molecular interactions between the Top1 linker and active site in mediating enzyme sensitivity to CPT are also supported by recent studies where the cytotoxicity induced by a defect in human Top1p-catalyzed DNA religation (due to the active site mutation T 722 A) was suppressed by the increased linker flexibility of the A 653 P mutation (32). Based on these considerations, we hypothesized that the presence of this conserved Gly residue within the active site of the enzyme (Gly 717 in human Top1p; Gly 721 in yeast Top1p) functions as a flexible hinge to facilitate the alterations in active site geometry and linker domain flexibility that impact CPT poisoning of Top1p.…”

Section: Discussionsupporting

confidence: 53%

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

Merwe

Bjornsti

2008

Journal of Biological Chemistry

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DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended ␣-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short ␣-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this ␣-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPTresistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.Eukaryotic DNA topoisomerase 1 (Top1p) 2 alters DNA topology by introducing a transient break in a single strand of the DNA duplex, thereby allowing strand rotation at the site of DNA scission to relieve superhelical tension (1-3). The enzyme plays critical roles in processes such as replication, transcription, recombination, and chromosomal condensation, with a reaction mechanism highly conserved from yeast to human. The active site tyrosine (Tyr 727 in yeast) acts as a nucleophile to cleave a single DNA strand, forming a covalent phosphotyrosyl linkage between the enzyme and the 3Ј-end of the DNA. The free 5Ј-OH end of the scissile strand then rotates about the uncleaved DNA strand in a manner dictated by torsional strain in the DNA. In a second transesterification reaction, the nucleophilic attack by the 5Ј-OH resolves the covalent Top1-DNA intermediate, and the enzyme is liberated from the religated DNA.DNA cleavage by Top1p is, at least in part, rate-limiting, thus ensuring low steady state-levels of the covalent enzyme-DNA complex (4). However, increased concentrations of covalent complexes, either as a consequence of drug action, Top1p mutation, or the formation of DNA adducts, converts Top1p into a cellular poison (5-16). The camptothecin (CPT) class of chemotherapeutics reversibly stabilizes the covalent intermediat...

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Section: Discussionsupporting

confidence: 53%

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

Merwe

Bjornsti

2008

Journal of Biological Chemistry

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“…Molecular dynamic simulations and x-ray crystallographic data suggest that restrictions in linker domain flexibility coincide with CPT binding to the Top1-DNA complex (11,23). Linker flexibility also impacts mutation-induced alterations in enzymecatalyzed DNA cleavage/religation (24). Indeed, our recent studies suggest that a conserved Gly within the catalytic pocket of Top1 provides a flexible hinge to facilitate linker mobility (26).…”

mentioning

confidence: 93%

“…Recently, we also demonstrated an interaction of the N-terminal and linker domains in regulating enzyme function in vivo. 5 Previous studies of Top1 reconstituted from independently expressed protein domains and the Top1A653P mutant indicate that the physical integrity and flexibility of the linker domain are critical determinants of enzyme sensitivity to CPT (20,(23)(24)(25). Molecular dynamic simulations and x-ray crystallographic data suggest that restrictions in linker domain flexibility coincide with CPT binding to the Top1-DNA complex (11,23).…”

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confidence: 99%

Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

Palle

Pattarello

Merwe

et al. 2008

Journal of Biological Chemistry

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In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions. Previously, reversible disulfide bonds were designed to assess whether locking the Top1 clamp around duplex DNA would restrict DNA strand rotation within the covalent Top1- To assess the contribution of the N-terminal domain to the dynamics of Top1 clamping of DNA, the same disulfide bonds were engineered into full-length Top1 and truncated Topo70, and the activities of these proteins were assessed in vitro and in yeast. Here we report that the N terminus impacts the opening and closing of the Top1 protein clamp. We also show that the architecture of yeast and human Top1 is conserved in so far as cysteine substitutions of the corresponding residues suffice to lock the Top1-clamp. However, the composition of the divergent N-terminal/linker domains impacts Top1-clamp activity and stability in vivo.Eukaryotic Top1 (DNA topoisomerase I) is a highly conserved enzyme, which plays critical roles in cellular processes, such as DNA replication, transcription, recombination, and chromosome condensation (1-4). Top1 catalyzes changes in DNA topology or the linkage of the individual strands that comprise duplex DNA. The enzyme first binds double-stranded DNA as a protein clamp and then transiently cleaves a single strand forming a 3Ј-phosphotyrosyl linkage between the active site tyrosine and the nicked DNA. Torsional or flexural strain in the DNA then drives the rewinding or unwinding of the polynucleotide strands. In the subsequent transesterification reaction, the free 5Ј-OH acts as a nucleophile to resolve the phosphotyrosyl linkage and religate the phosphodiester backbone of the DNA. A random number of supercoils are removed in this process to effect changes in DNA linking number (5, 6).Top1 is also the cellular target of the camptothecin (CPT) 4 class of chemotherapeutic agents (3, 7-9). These drugs convert the enzyme into a cellular poison by reversibly stabilizing the Top1-DNA covalent complex. CPT intercalates into the protein-linked DNA complex, effectively displacing the 5Ј-OH and preventing DNA religation (10, 11). Although ternary CPTTop1-DNA complexes form throughout the cell cycle, the interaction of these complexes with the replication machinery during S-phase converts the readily reversible intermediate into irreversible DNA lesions that trigger cell death. Our recent in vivo studies and single molecule nanomanipulations revealed that CPT selectively slows Top1 uncoiling of positive supercoils, thereby inducing the accumulation of ...

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“…YCp-GAL1-hTOP1G363C, YCpGAL1-hTOP1K720E, and YCp-GAL1-hTOP1A653P plasmids express the mutant (G363C, K720E, and A653P) form of the human DNA topoisomerase I under the control of galactose-inducible GAL1 promoter. Yeast cells transformed by treatment with lithium acetate (16) were maintained at 30jC in synthetic complete medium lacking uracil (uracil-) and supplemented with 2% glucose. For yeast spot test, cells were growth at 30jC in uracil-medium to an A 595 of 0.3.…”

Section: Methodsmentioning

confidence: 99%

Preclinical profile of antitumor activity of a novel hydrophilic camptothecin, ST1968

Pisano

Cesare

Beretta

et al. 2008

Molecular Cancer Therapeutics

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ST1968 is a novel hydrophilic camptothecin (CPT) derivative of the 7-oxyiminomethyl series. Because ST1968 retained ability to form remarkably stable cleavable complexes, this study was done to investigate its preclinical profile of antitumor activity in a large panel of human tumor models, including irinotecan-resistant tumors. Although less potent than SN38 in vitro, i.v. administered ST1968 caused a marked tumor inhibition, superior to that of irinotecan, in most tested models. ST1968 exhibited an impressive activity against several tumors including models of ovarian and colon carcinoma in which a high rate of cures was observed. In the most responsive tumors, complete and persistent tumor regressions were achieved even with low suboptimal doses. Even tumors derived from intrinsically resistant cells exhibited a significant responsiveness.

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Alterations in Linker Flexibility Suppress DNA Topoisomerase I Mutant-induced Cell Lethality

Cited by 24 publications

References 40 publications

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

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

Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

Preclinical profile of antitumor activity of a novel hydrophilic camptothecin, ST1968

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