The Experts below are selected from a list of 23649 Experts worldwide ranked by ideXlab platform
James C Wang - One of the best experts on this subject based on the ideXlab platform.
-
protein footprinting at cysteines probing atp modulated contacts in cysteine substitution mutants of yeast DNA Topoisomerase ii
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Benjamin P Tu, James C WangAbstract:Cysteine-substitution mutants of yeast DNA Topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA Topoisomerase II.
-
reconstitution of DNA Topoisomerase vi of the thermophilic archaeon sulfolobus shibatae from subunits separately overexpressed in escherichia coli
Nucleic Acids Research, 1998Co-Authors: Cyril Buhler, Daniele Gadelle, Patrick Forterre, James C Wang, Agnes BergeratAbstract:DNA Topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA Topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA Topoisomerases and archaeal DNA gyrases. DNA Topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA Topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.
-
moving one DNA double helix through another by a type ii DNA Topoisomerase the story of a simple molecular machine
Quarterly Reviews of Biophysics, 1998Co-Authors: James C WangAbstract:. . Transporting one DNA double helix through another . The clamp model: a type II DNA Topoisomerase as an ATP-modulated protein clamp . The number of gates in the protein clamp . Three-dimensional structures of type II DNA Topoisomerase fragments . How are the various subfragments connected? . A molecular model of DNA transport by a type II DNA Topoisomerase
-
structure and mechanism of DNA Topoisomerase ii
Nature, 1996Co-Authors: Steven J Gamblin, James M Berger, Stephen C Harrison, James C WangAbstract:The crystal structure of a large fragment of yeast type II DNA Topoisomerase reveals a heart-shaped dimeric protein with a large central hole. It provides a molecular model of the enzyme as an ATP-modulated clamp with two sets of jaws at opposite ends, connected by multiple joints. An enzyme with bound DNA can admit a second DNA duplex through one set of jaws, transport it through the cleaved first duplex, and expel it through the other set of jaws.
-
the probabilities of supercoil removal and decatenation by yeast DNA Topoisomerase ii
Genes to Cells, 1996Co-Authors: Joaquim Roca, James C WangAbstract:Background: In yeast a single type II DNA Topoisomerase is involved in both the removal of DNA supercoils and the unlinking of intertwined pairs of newly replicated chromosomes or plasmids; in bacteria, two type II enzymes, DNA gyrase and DNA Topoisomerase IV, function separately in the passage of DNA segments in cis and in trans. To deduce the molecular characteristics of these enzyme-mediated reactions, the efficiencies of supercoil removal and decatenation by the yeast enzyme upon the addition of a nonhydrolysable ATP analogue were determined. Results: The probability that a bound enzyme transports a DNA segment in cis increases with positive or negative supercoiling of the DNA, and transport is nearly quantitative at high degrees of supercoiling. The relative probabilities of transporting a contiguous and noncontiguous DNA segment by a yeast enzyme bound to one member of a singly linked pair of 3.6-kb rings were calculated from the observed efficiency of decatenation. When the enzyme-bound ring is highly supercoiled, transport of a noncontiguous segment is more probable than a contiguous one. Conclusion: A DNA-bound yeast enzyme has no intrinsic bias in its selection of a contiguous or noncontiguous DNA segment for transport, and the selection is determined by DNA conformations. For the singly linked dimeric catenane studied, a high degree of supercoiling of the enzyme-bound DNA does not make supercoil removal more favourable than decatenation. In the case of bacterial gyrase, however, wrapping of a DNA segment around the enzyme is expected to strongly favour the transport of a contiguous segment.
Ryuichi Okayasu - One of the best experts on this subject based on the ideXlab platform.
-
DNA Topoisomerase inhibitor etoposide enhances gc box dependent promoter activity via sp1 phosphorylation
Cancer Science, 2007Co-Authors: Ichiro Niina, Takayuki Torigoe, Tomonori Igarashi, Tetsuro Wakasugi, Naoya Miyamoto, Tetsuro Onitsuka, Hiroto Izumi, Masaki Shiota, Takeshi Uchiumi, Ryuichi OkayasuAbstract:Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA Topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA Topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance. (Cancer Sci 2007; 98: 858–863)
-
DNA Topoisomerase inhibitor etoposide enhances gc box dependent promoter activity via sp1 phosphorylation
Cancer Science, 2007Co-Authors: Ichiro Niina, Takayuki Torigoe, Tomonori Igarashi, Tetsuro Wakasugi, Naoya Miyamoto, Hiroto Izumi, Masaki Shiota, Takeshi Uchiumi, Takamitsu Onitsuka, Ryuichi OkayasuAbstract:Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA Topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA Topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance.
Patrick Forterre - One of the best experts on this subject based on the ideXlab platform.
-
Crystal structure of an intact type II DNA Topoisomerase: insights into DNA transfer mechanisms.
Structure Struct Fold Des; Structure (Camb ), 2008Co-Authors: Marc Graille, Daniele Gadelle, Patrick Forterre, Lionel Cladière, Denys Durand, François Lecointe, Sophie Quevillon-cheruel, Patrice Vachette, Herman Van TilbeurghAbstract:DNA Topoisomerases resolve DNA topological problems created during transcription, replication, and recombination. These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Type II enzymes catalyze the transfer of a DNA duplex through another one in an ATP-dependent mechanism. Because of its small size and sensitivity to antitumoral drugs, the archaeal DNA Topoisomerase VI, a type II enzyme, is an excellent model for gaining further understanding of the organization and mechanism of these enzymes. We present the crystal structure of intact DNA Topoisomerase VI bound to radicicol, an inhibitor of human topo II, and compare it to the conformation of the apo-protein as determined by small-angle X-ray scattering in solution. This structure, combined with a wealth of experimental data gathered on these enzymes, allows us to propose a structural model for the two-gate DNA transfer mechanism.
-
the hsp90 and DNA Topoisomerase vi inhibitor radicicol also inhibits human type ii DNA Topoisomerase
Biochemical Pharmacology, 2006Co-Authors: Daniele Gadelle, Patrick Forterre, Marc GrailleAbstract:Radicicol derivatives are currently investigated as promising antitumoral drugs because they inhibit the activity of the molecular chaperone heat shock protein (HSP90), causing the destabilization and eventual degradation of HSP90 client proteins that are often associated with tumor cells. These drugs interact with the ATP-binding site of HSP90 which is characterized by a structural element known as the Bergerat fold, also present in type II DNA Topoisomerases (Topo II). We have previously shown that radicicol inhibits archaeal DNA Topoisomerase VI, the prototype of Topo II of the B family (present in archaea, some bacteria and all the plants sequenced so far). We show here that radicicol also inhibits the human Topo II, a member of the A family (comprising the eukaryotic Topo II, bacterial gyrase, Topo IV and viral Topo II), which is a major target for antitumoral drugs. In addition, radicicol prevents in vitro induction of DNA cleavage by human Topo II in the presence of the antitumoral drug etoposide. The finding that radicicol can inhibit at least two different antitumoral drug targets in human, and interferes with drugs currently used in cancer treatment, could have implications in cancer therapy.
-
inhibition of archaeal growth and DNA Topoisomerase vi activities by the hsp90 inhibitor radicicol
Nucleic Acids Research, 2005Co-Authors: Daniele Gadelle, C Bocs, Marc Graille, Patrick ForterreAbstract:Type II DNA Topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA Topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA Topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA Topoisomerase VI ‘Bergerat fold’, whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA Topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.
-
reconstitution of DNA Topoisomerase vi of the thermophilic archaeon sulfolobus shibatae from subunits separately overexpressed in escherichia coli
Nucleic Acids Research, 1998Co-Authors: Cyril Buhler, Daniele Gadelle, Patrick Forterre, James C Wang, Agnes BergeratAbstract:DNA Topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA Topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA Topoisomerases and archaeal DNA gyrases. DNA Topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA Topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.
Ichiro Niina - One of the best experts on this subject based on the ideXlab platform.
-
DNA Topoisomerase inhibitor etoposide enhances gc box dependent promoter activity via sp1 phosphorylation
Cancer Science, 2007Co-Authors: Ichiro Niina, Takayuki Torigoe, Tomonori Igarashi, Tetsuro Wakasugi, Naoya Miyamoto, Tetsuro Onitsuka, Hiroto Izumi, Masaki Shiota, Takeshi Uchiumi, Ryuichi OkayasuAbstract:Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA Topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA Topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance. (Cancer Sci 2007; 98: 858–863)
-
DNA Topoisomerase inhibitor etoposide enhances gc box dependent promoter activity via sp1 phosphorylation
Cancer Science, 2007Co-Authors: Ichiro Niina, Takayuki Torigoe, Tomonori Igarashi, Tetsuro Wakasugi, Naoya Miyamoto, Hiroto Izumi, Masaki Shiota, Takeshi Uchiumi, Takamitsu Onitsuka, Ryuichi OkayasuAbstract:Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA Topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA Topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance.
Rosa M Reguera - One of the best experts on this subject based on the ideXlab platform.
-
a pentapeptide signature motif plays a pivotal role in leishmania DNA Topoisomerase ib activity and camptothecin sensitivity
Biochimica et Biophysica Acta, 2012Co-Authors: Christopher F Prada, Rafael Balanafouce, Yolanda Perezpertejo, Raquel Alvarezvelilla, Rosario Diazgonzalez, C Prieto, Rosa M RegueraAbstract:Abstract Background Leishmania donovani – the causative agent of visceral leishmaniasis – has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA Topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania . Methods Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin. Results Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA Topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1–592 and 175–262 in the large and small subunits, respectively. Conclusion It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania . General significance The present report describes a functional analysis of the leishmanial DNA Topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.
-
gene disruption of the DNA Topoisomerase ib small subunit induces a non viable phenotype in the hemoflagellate leishmania major
BMC Microbiology, 2008Co-Authors: Rafael Balanafouce, Carlos Garciaestrada, Yolanda Perezpertejo, Rosa M RegueraAbstract:Background The unusual heterodimeric leishmanial DNA Topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate Trypanosoma brucei. Unfortunately, this approach is not suitable in Leishmania where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the L. major DNA Topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.
-
gene disruption of the DNA Topoisomerase ib small subunit induces a non viable phenotype in the hemoflagellate leishmania major
BMC Microbiology, 2008Co-Authors: Rafael Balanafouce, Carlos Garciaestrada, Yolanda Perezpertejo, Rosa M RegueraAbstract:The unusual heterodimeric leishmanial DNA Topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate Trypanosoma brucei. Unfortunately, this approach is not suitable in Leishmania where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the L. major DNA Topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively. We have successfully replaced both topS loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the Leishmania major topS gene. Phenotypic characterization of the L. major rescued strain and a L. major strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in L. major, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G2/M reversible arrest was the main outcome at lower concentrations, but irreversible G2 arrest was detected at higher camptothecin pressure. Results obtained in this work evidence the essentiality of the topS gene encoding the L. major DNA Topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in Leishmania parasites.