Structure and Hydration of the DNA-Human Topoisomerase I Covalent Complex

Chillemi, Giovanni; Castrignanò, Tiziana; Desideri, Alessandro

doi:10.1016/s0006-3495(01)75716-5

Cited by 30 publications

(45 citation statements)

References 47 publications

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“…Two MD simulation studies have indicated the occurrence of long range communications between different topoisomerase domains (33,34). A specific long range communication, involving the drug binding region, the geometry of the active site, and the linker domain has been demonstrated by the differential detection of the linker domain through x-ray diffraction only in the topoisomerase-DNA complex in the presence of Topotecan (8).…”

Section: Effect Of Cpt On the Cleavage-religation Equilibrium-tomentioning

confidence: 99%

Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Fiorani

Bruselles

Falconi

et al. 2003

Journal of Biological Chemistry

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DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance. DNA topoisomerase I (Top1p)1 catalyzes the relaxation of supercoiled DNA through the transient cleavage of one strand of a DNA duplex and is fundamental to processes such as replication, recombination, and transcription (1-4). The threedimensional structure of reconstituted and N-terminal truncated versions of human topoisomerase I (hTop1p) in complex with a 22-bp DNA molecule have shown that the enzyme is organized in multiple domains that "clamp" around the DNA molecule (5, 6). Changes in DNA topology are achieved by introducing a transient break in the phosphodiester bond of one strand in the duplex DNA. The phosphodiester bond energy is preserved during catalysis through the formation of a transient covalent phosphotyrosine bond between the catalytic Tyr-723 and the 3Ј end of the broken DNA strand. DNA relaxation has been proposed to proceed via "controlled rotation" in which the covalently bound enzyme holds one end of the DNA duplex and allows the end downstream of the cleavage site to rotate around the remaining phosphodiester bond (6). Eukaryotic Top1p is the cellular target of the anti-tumor drug camptothecin (CPT), which reversibly stabilizes the cleavable intermediate complex formed in the catalytic cycle of the enzyme (1, 2, 7).Recently Staker et al. (8) have solved the x-ray crystal structure of hTop1p covalently joined to double-stranded DNA and bound to the CPT analog Topotecan (8). This structure revealed that the drug molecule intercalates between upstream (Ϫ1) and downstream (ϩ1) bp, displacing the downstream DNA and thus preventing the religation of the cleaved strand. The structure helps to clarify the role of several, but not all, mutant residues that produce a CPT-resistant enzyme. In particular the structure permits to explain the CPT resistance for mutations involving residues that interact directly with the drug or that would alter the interaction with DNA...

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Section: Effect Of Cpt On the Cleavage-religation Equilibrium-tomentioning

confidence: 99%

Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Fiorani

Bruselles

Falconi

et al. 2003

Journal of Biological Chemistry

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“…The following modifications were made to this structure: (i) the iodouracils were converted to thymines; (ii) the crystallographic water molecules, except for the long-life [30] bridging water doubly hydrogen bonded to protonated Arg488 and ionized Asp533, were removed; (iii) hydrogen atoms were added to both the DNA and the enzyme; and (iv) the entire complex was energy minimized using the Tripos force field to the conjugate gradient of 1 kcal/(mol Å ).…”

Section: Construction Of the Ternary Complexesmentioning

confidence: 99%

Human topoisomerase I poisoning: docking protoberberines into a structure-based binding site model

Kettmann

Kostálová

Höltje

2004

J Comput Aided Mol Des

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Using the X-ray crystal structure of the human topoisomerase I (top1) - DNA cleavable complex and the Sybyl software package, we have developed a general model for the ternary cleavable complex formed with four protoberberine alkaloids differing in the substitution on the terminal phenyl rings and covering a broad range of the top1-poisoning activities. This model has the drug intercalated with its planar chromophore between the -1 and +1 base pairs flanking the cleavage site, with the nonplanar portion pointing into the minor groove. The ternary complexes were geometry-optimized and relative interaction energies, computed by using the Tripos force field, were found to rank in correct order the biological potency of the compounds; in addition, the model is also consistent with the top1-poisoning inactivity of berberine, a major prototype of the protoberberine alkaloids. The model might serve as a rational basis for elaboration of the most active compound as a lead structure, in order to develop more potent top1 poisons as next generation anti-cancer drugs.

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“…Both these regions have been shown to form several direct and water-mediated contacts with the DNA phosphate groups in the strands downstream of the scissile base (5,11,13). It is worth noting, therefore, that the greater changes of dynamics and structure, in the absence of DNA but in the closed conformation, are located in the two regions involved in the controlling of the rotation of the scissile strand during the relaxation step.…”

Section: Resultsmentioning

confidence: 99%

“…Starting from the hTop1 closed crystallographic structure, the flexibility of the different domains have been monitored, but it has not been possible to monitor the so-called open state conformation, because of the strict non-covalent interactions occurring between the protein domains and DNA (11). MD can also be applied to sample large conformational changes after introducing a perturbation or a force that imposes the system to sample events occurring on a timescale not accessible by the current computational resources.…”

Section: Introductionmentioning

confidence: 99%

The open state of human topoisomerase I as probed by molecular dynamics simulation

Chillemi

Bruselles

Fiorani

et al. 2007

Nucleic Acids Research

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The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80–90 Å, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7 Å. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.

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Structure and Hydration of the DNA-Human Topoisomerase I Covalent Complex

Cited by 30 publications

References 47 publications

Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Single Mutation in the Linker Domain Confers Protein Flexibility and Camptothecin Resistance to Human Topoisomerase I

Human topoisomerase I poisoning: docking protoberberines into a structure-based binding site model

The open state of human topoisomerase I as probed by molecular dynamics simulation

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