Role of the Linker Domain and the 203–214 N-Terminal Residues in the Human Topoisomerase I DNA Complex Dynamics

Chillemi, Giovanni; Redinbo, Matthew R.; Bruselles, Alessandro; Desideri, Alessandro

doi:10.1529/biophysj.104.044925

Cited by 24 publications

(28 citation statements)

References 53 publications

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“…The identical religation rate of the wild-type and the mutated enzyme is actually an unexpected result since it has been suggested that Thr-718 plays a role in the right positioning of the 5′ end through a hydrogen bond between the aminoacidic hydroxyl group and the G+2 phosphate group of the scissile strand (8). The MD trajectory confirms the importance of this hydrogen bond since it is present in the native enzyme for the entire duration of the simulation (30). However, the simulation also shows that upon mutation of the threonine in alanine the lack of the direct hydrogen bond with the G+2 phosphate group is compensated by a water-mediated hydrogen bond to the backbone of Ala-718 (Figure 8).…”

Section: Discussionsupporting

confidence: 60%

“…This suggests that the main effect caused by the mutation as detected by experiments carried out at 150 mM KCl ionic strength is a perturbation in the dynamical behavior that alters the control of the DNA strand rotation. In order to sample the dynamical properties of the mutated enzyme, to be correlated with the above reported experimental findings, we have carried out a 3250 ps long MD simulation of the mutated system, and compared it with an identical simulation carried out on the native enzyme (30). …”

Section: Resultsmentioning

confidence: 99%

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Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

Chillemi¹

2005

Nucleic Acids Research

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The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped α helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle.

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Section: Discussionsupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

Chillemi¹

2005

Nucleic Acids Research

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“…Staker et al posited that this interaction might position the 5Ј-OH end of the cleaved DNA for nucleophilic attack of the 3Ј-phosphotyrosyl bond to effect DNA religation (17); thereby providing an explanation for the lethal phenotype of the T718A mutant (10,12,15). Indeed, recent molecular dynamic simulations of the Topo70-DNA complex further support the formation of a hydrogen bond between Thr 718 and the G ϩ2 phosphate group, consistent with a model whereby enzyme controls the relative position of the ϩ1 base with regard to the active site, by means of hydrogen bonds with the ϩ2 G phosphate group (44). Taken together, these observations indicate that the two mutations A653P and T718A induce opposite effects on enzyme catalysis: A653P enhances (21), whereas T718A decreases DNA religation (14,16).…”

Section: Discussionmentioning

confidence: 59%

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

Losasso

Cretaio

Palle

et al. 2007

Journal of Biological Chemistry

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Human DNA topoisomerase I (Top1p) 3 plays a critical role in processes such as replication, recombination, and transcription (1-4). This 91-kDa enzyme catalyzes the relaxation of supercoiled DNA through the transient cleavage of one strand of the DNA duplex. The Top1p catalytic cycle comprises five sequential steps: DNA binding, cleavage, strand rotation, religation, and release (1-4). During enzyme catalysis, Top1p undergoes large conformational changes, from an "open" structure that allows DNA binding, to the "closed clamp" conformation observed in co-crystal structures, in which the enzyme completely embraces the duplex DNA (5). DNA relaxation appears to proceed via a mechanism of DNA strand rotation, where the covalent attachment of Top1p to the 3Ј-phosphoryl end of the nicked DNA strand liberates the 5Ј-OH end to rotate around the intact nonscissile DNA strand (6). Remarkably, the dynamic changes in protein clamp conformation and linkage of DNA strands occur in the absence of any energy cofactors, such as ATP.Eukaryotic DNA topoisomerase I is the cellular target of the antitumor drug camptothecin (CPT) (7-9). CPT specifically and reversibly binds the covalent Top1p-DNA complex to inhibit DNA religation and induce S-phase-dependent cell lethality. In the yeast Saccharomyces cerevisiae, single amino acid substitutions have been defined in Top1p that mimic the cytotoxic activity of CPT by inducing a terminal G 2 -phase phenotype and increased rates of recombination (10 -14). For example, substitution of Ala for residue Thr 722 (within the active site Tyr 727 domain) induces a dramatic reduction in cell viability when expressed in yeast or human cells (10,15). Similar to the action of CPT, this mutation enhances the stability of covalent enzyme-DNA complexes by a reduction in the rate of DNA religation (14). Substitution of Ala for the corresponding residue Thr 718 of human Top1p (top1T718A) induces a similar phenotype and alterations in enzyme catalysis (12). Thus, in yeast and human Top1p, mutation of this conserved Thr residue (located just five residues N-terminal to the active site Tyr) induces cell lethality as a consequence of an increase in the half-life of the covalent enzyme-DNA intermediate (14,16). Recent structures of the C-terminal 70-kDa fragment of human Top1p (Topo70) with DNA and the ternary topotecan Topo70-DNA complex indicate the formation of a hydrogen bond between the hydroxyl of Thr 718 and the nonbridging oxygen of the ϩ1 phosphodiester of the DNA (17). These data suggest that substitution of Ala in this position may alter the orientation of the 5Ј-OH nucleophile to alter the rate of DNA religation.Structural and biochemical data indicate DNA topoisomerase I contains multiple domains that form a protein clamp around duplex DNA (18) (see Fig. 1). Based on limited proteol-* This work was supported in part by grants from the Ministero dell'Instruzione, dell'Università e della Ricerca Cofinanziamento, FIRB, Ministero della Salute, and Genomica Funzionale CNR (to P. B.), the National Institu...

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“…4B). Biochemical and molecular modeling studies indicate that the linker domain influences cleavage-religation dynamics and equilibrium (14,(29)(30)(31). Structural homology modeling and secondary structure prediction (32,33) suggest that the Top1mt linker domain is less structured and presumably more flexible than the nTop1 linker domain.…”

Section: Discussionmentioning

confidence: 99%

A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

Seol

Zhang

Pommier

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Type IB topoisomerases (Top1Bs) relax excessive DNA supercoiling associated with replication and transcription by catalyzing a transient nick in one strand to permit controlled rotation of the DNA about the intact strand. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Top1B (nTop1). Previous work on vaccinia Top1B led to an elegant model that describes a straightforward dependence of rotation and religation on the torque caused by supercoiling. Here, we used a singlemolecule DNA supercoil relaxation assay to measure the torque dependence of nTop1 and its inhibition by CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTop1. Despite substantial sequence homology in their core domains, nTop1 and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and CPT, with the N-terminal deletion mutant of nTop1 showing intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torqueindependent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation and the efficiency of CPT binding. This "kinetic clutch" mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.cancer therapy | magnetic tweezers | single-molecule biophysics T ype IB topoisomerase (Top1B) is essential for relaxing DNA supercoils generated during replication, transcription, DNA recombination, and repair (1-4). Due to its prominent roles in DNA metabolism, human Top1B is an important target for cancer therapies (2, 4, 5). Top1B relaxes DNA supercoils by forming a clamp around the DNA and generating a transient nick, which allows rotation of the DNA until the nick is religated (6, 7). This DNA rotation was thought to be hindered by interactions with the protein clamp because the cross-section of rotating DNA at the nick increases twofold during rotation (6,8,9). This was confirmed in a pioneering single-molecule study of relaxation by vaccinia Top1B that led to a model of DNA rotation as a random walk along a rugged energy landscape associated with the rotation angle of the 5′ end of the DNA. In this model, the rotation rate is dictated by the escape from multiple energy wells; religation is in kinetic competition with rotation at a specific energy well. Torque on the DNA (i.e., supercoiling) tilts the energy landscape, lowering the rotation energy barriers, thereby increasing the rotation rate and decreasing the probability of religation. As a result, both the rotation rate and the religation probability are torque-dependent to different degrees. This simple yet elegant model describes vaccinia Top1B supercoil relaxation as a fu...

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Role of the Linker Domain and the 203–214 N-Terminal Residues in the Human Topoisomerase I DNA Complex Dynamics

Cited by 24 publications

References 53 publications

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

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

A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

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