PubMed Health⌕ Search

Biomedical subjects

Y C Tse-Dinh

Publications and source records attributed to Y C Tse-Dinh.

At least 19 recordsLinked to original sources
PubMed2001

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I.

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.

Binding Sites↗
PubMed2001

Evaluation of topotecan and etoposide for non-Hodgkin lymphoma: correlation of topoisomerase-DNA complex formation with clinical response.

BACKGROUND: Topotecan, a topoisomerase I inhibitor, acts by stabilizing the topoisomerase DNA cleavage complex. Etoposide, a topoisomerase II inhibitor, mediates antitumor activity by stabilizing cleavage complex formed between topoisomerase II and DNA. These two agents have therapeutic activity in non-Hodgkin lymphoma. The authors report Phase I data of topotecan and etoposide combination for patients with recurrent or refractory non-Hodgkin lymphoma and correlation of topoisomerase-DNA complex formation to clinical response. METHODS: Twenty-two patients with recurrent or refractory aggressive non-Hodgkin lymphoma were treated at four dose levels of topotecan (1 mg/m(2)/day to 2.5 mg/m(2)/day). Topotecan was given at a 30-minute infusion daily with etoposide 150 mg/m(2)/day, both for 5 days. Topoisomerase-DNA covalent complex formation was measured using in vivo link assay, whereas topoisomerase I, IIalpha, and IIbeta in RNA expression levels were determined by reverse transcription-polymerase chain reaction in blood samples. The relation of these levels to clinical response was studied. RESULTS: The maximum tolerated dose of topotecan was 2.0 mg/m(2)/day for 5 days. Oropharyngeal mucositis was dose-limiting. Of 21 examinable patients, 3 patients achieved complete remission, and 5 patients achieved partial remission. Of six untreated patients who experienced a recurrence, three had complete remission, and the other three had partial remission. Drug-induced topoisomerase-DNA complex formation was observed throughout the treatment in blood samples of all the patients who responded. However, only 4 of 13 patients, who did not respond, formed covalent complex at all time points. This was statistically significant (P = 0.024). In all patients, expression levels of topoisomerase I and IIbeta mRNA remained similar to pretreatment levels, whereas topoisomerase IIalpha mRNA levels decreased dramatically by the third day. CONCLUSION: The recommended Phase II dose of topotecan with etoposide of 150 mg/m(2)/day for 5 days was 2.0 mg/m(2)/day for 5 days. Topoisomerase-DNA complex formation correlated with response to treatment.

Adult↗
PubMed2000

Differential expression of human topoisomerase IIIalpha during the cell cycle progression in HL-60 leukemia cells and human peripheral blood lymphocytes.

Human topoisomerase IIIalpha (huTop IIIalpha) has been demonstrated to belong to type IA subfamily. In this study, we found that huTop IIIalpha expressed constitutively and remained at high levels throughout the cell cycle in HL-60 cells when compared to the cell-cycle-dependent expression of huTop IIIalpha in phytohemagglutinin-activated peripheral blood lymphocytes. During the cell cycle progression, this protein remained accentuated in the nucleolus without significant translocation from the nucleolus to the nucleoplasm. In addition, during the course of granulocytic maturation in DMSO-treated HL-60 cells, huTop IIIalpha levels decreased when cells stopped proliferation and nucleoli diminished in size. However, its level remained unchanged during the course of monocytic maturation of vitamin D(3)-treated HL-60 cells which still retained its proliferative capacity and did not change the size of the nucleolus. The data suggested that huTop IIIalpha is involved in rDNA metabolism, such as rDNA transcription. Its cellular level appeared to be under control during the cell cycle progression of normal lymphocytes, but was found to be deregulated in HL-60 cells which may be associated with the tumor transformed cell phenotypes.

Amino Acid Sequence↗
PubMed2000

The acidic triad conserved in type IA DNA topoisomerases is required for binding of Mg(II) and subsequent conformational change.

The acidic residues Asp-111, Asp-113, and Glu-115 of Escherichia coli DNA topoisomerase I are located near the active site Tyr-319 and are conserved in type IA topoisomerase sequences with counterparts in type IIA DNA topoisomerases. Their exact functional roles in catalysis have not been clearly defined. Mutant enzymes with two or more of these residues converted to alanines were found to have >90% loss of activity in the relaxation assay with 6 mM Mg(II) present. Mg(II) concentrations (15-20 mM) inhibitory for the wild type enzyme are needed by these double mutants for maximal relaxation activity. The triple mutant D111A/D113A/E115A had no detectable relaxation activity. Mg(II) binding to wild type enzyme resulted in an altered conformation detectable by Glu-C proteolytic digestion. This conformational change was not observed for the triple mutant or for the double mutant D111A/D113A. Direct measurement of Mg(II) bound showed the loss of 1-2 Mg(II) ions for each enzyme molecule due to the mutations. These results demonstrate a functional role for these acidic residues in the binding of Mg(II) to induce the conformational change required for the relaxation of supercoiled DNA by the enzyme.

Alanine↗
PubMed1999

Increased thermosensitivity associated with topoisomerase I deletion and promoter mutations in Escherichia coli.

An Escherichia coli mutant with three of the promoters for the topoisomerase I gene (topA) deleted, such that only the sigma 32-dependent promoter (P1) remained, had a decreased level of topoisomerase I at 30 degrees C and showed increased thermosensitivity at 52 degrees C. However, it could still develop thermotolerance and had a wild-type level of resistance to 52 degrees C treatment if exposed first to 42 degrees C. This indicated that newly synthesized topoisomerase I from transcription initiated at P1 was important for development of thermotolerance. Two other E. coli mutants lacking topA were > 100 times more sensitive to high temperature than their wild-type isogenic strains.

DNA Topoisomerases, Type I↗
PubMed1999

Microbial resistance: novel screens for a contemporary problem.

Historically, natural products have been the source of a large variety of antibacterial agents. In the 1980s, no additional useful antibacterial agents were discovered, leading to the belief that most useful chemotypes from natural product sources had already been discovered. At this time, advances in biotechnology made it feasible to produce sufficient enzyme to set up cell-free screens. Chemical compound libraries and combinatorial synthesis became the source of chemical diversity for the screens. In spite of these efforts, very few new antibacterial agents have been discovered in the last decade. At Small Molecule Therapeutics, Inc., we have developed phenotype-based screens that take advantage of the natural physiology and biochemistry of the target enzymes. We have developed a screen to identify bacterial DNA gyrase and topoisomerase IV poisons. The "hits" identified in this screen are being characterized further. A second screen has also been developed against bacterial topoisomerase 1 in which compounds that cause DNA damage through their interaction with bacterial topoisomerase 1 have been identified. Three of the compounds identified in the screen inhibit DNA relaxation mediated by bacterial topoisomerase 1, induce DNA cleavage, are noncytotoxic at >10 microM, and have MICs of 4.0 microg/mL against Staphylococcus aureus.

Animals↗
PubMed1998

The Zn(II) binding motifs of E. coli DNA topoisomerase I is part of a high-affinity DNA binding domain.

Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs. Three subclones containing these tetracysteine motifs were expressed and purified. Subclone ZD1 contained the minimal tetracysteine motifs sequence. A larger subclone ZD2 corresponded to a region bordered by two protease sensitive sites. Subclone ZD3 also included the 14-kDa C-terminal domain that has been shown to bind DNA. Subclones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity. ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain. The complex formed between ZD3 and a single-stranded 31mer could be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis conditions. The three Zn(II) binding motifs appeared to be part of a high-affinity DNA binding domain.

Amino Acid Sequence↗
PubMed1998

Bacterial and archeal type I topoisomerases.

Bacterial and archeal type I topoisomerases, including topoisomerase I, topoisomerase III and reverse gyrase, have different potential roles in the control of DNA topology including regulation of supercoiling and maintenance of genetic stability. Analysis of their coding sequences in different organisms shows that they belong to the type IA family of DNA topoisomerases, but there is variability in organization of various enzymatic domains necessary for topoisomerase activity. The torus-like structure of the conserved transesterification domain with the active site tyrosine for DNA cleavage/rejoining suggests steps of enzyme conformational change driven by DNA substrate and Mg(II) cofactor binding, that are required for catalysis of change in DNA linking number.

Archaeal Proteins↗
PubMed1998

Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I.

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42 degreesC, possibly accounting for the conservation of these residues in evolution.

Amino Acid Sequence↗
PubMed1997

Effect of Mg(II) binding on the structure and activity of Escherichia coli DNA topoisomerase I.

Escherichia coli DNA topoisomerase I requires Mg(II) as a cofactor for the relaxation of negatively supercoiled DNA. Mg(II) binding to the enzyme was shown by fluorescence spectroscopy to affect the tertiary structure of the enzyme. Addition of 2 mM MgCl2 resulted in a 30% decrease in the maximum emission of tryptophan fluorescence of the enzyme. These Mg(II)-induced changes in fluorescence properties were reversible by the addition of EDTA and not obtained with other divalent cations. After incubation with Mg(II) and dialysis, inductively coupled plasma (ICP) analysis showed that each enzyme molecule could form a complex with 1-2 Mg(II) bound to each enzyme molecule. Such Mg(II).enzyme complexes were found to be active in the relaxation of negatively supercoiled DNA in the absence of additional Mg(II). Results from ICP analysis after equilibrium dialysis and relaxation assays with limiting Mg(II) concentrations indicated that both Mg(II) binding sites had to be occupied for the enzyme to catalyze relaxation of negatively supercoiled DNA.

DNA Topoisomerases, Type I↗
PubMed1997

Regulation of Escherichia coli topA gene transcription: involvement of a sigmaS-dependent promoter.

To investigate the regulation of Escherichia coli topA gene transcription, primer extension was employed to determine the transcription initiation sites from the chromosomal topA gene. When cells were grown in LB medium to log phase, four transcription initiation sites could be identified. Three of these sites corresponded to promoters P1, P2 and P4 previously characterized using topA-galK fusion plasmids. The P3 promoter that is active on the plasmid was not utilized at the chromosomal topA gene under the conditions employed. There was a new transcription initiation site corresponding to a new promoter Px1. When cells started to enter stationary phase, promoter Px1 gradually became the major transcription initiation site for topA, while transcription from promoters P2 and P4 decreased. In an E. coli mutant lacking sigmaS (the rpoS gene product), the stationary phase specific sigma factor, the induction of transcription from promoter Px1 was abolished. In another mutant lacking H-NS activity, resulting in increased sigmaS level in log-phase, the transcription from promoter Px1 during log phase was increased. Thus Px1 appeared to be regulated by sigmaS. The activity of promoter P1 on the chromosome increased during heat shock, consistent with the previous result obtained using the topA-galK fusion plasmid showing that P1 is a sigma32-dependent heat shock promoter. Promoters P2 and P4 were most likely to be recognized by sigma70. The total level of topoisomerase I protein in the rpoS mutant was not reduced significantly in stationary phase due to increased transcription initiation from the other topA promoters. The utilization of multiple sigma factors for transcription initiation of topA could be important for adaptation of E. coli to change in growth conditions.

Bacterial Proteins↗
PubMed1997

DNA supercoiling and bacterial adaptation: thermotolerance and thermoresistance.

When bacterial cells are shifted to higher temperatures their degree of DNA supercoiling changes. Topoisomerases are involved in bacterial adaptation to environmental changes requiring rapid shifts in gene expression. This role in heat shock has been elucidated by genetic studies on the Escherichia coli topA gene and its sigma 32-dependent promoter, P1. Other studies have shown that certain gyrA mutants have increased thermoresistance.

Adaptation, Physiological↗
PubMed1996

Backbone dynamics of the C-terminal domain of Escherichia coli topoisomerase I in the absence and presence of single-stranded DNA.

The backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

Base Sequence↗
PubMed1996

Vaccinia virus DNA topoisomerase I preferentially removes positive supercoils from DNA.

Type I DNA topoisomerases homologous to Escherichia coli topoisomerase I normally only remove negative supercoils from DNA. Topoisomerases I from various eukaryotes share sequence homology and remove both positive and negative supercoils from DNA. Here we report that vaccinia virus topoisomerase I has significant difference in substrate preference from the other homologous type I topoisomerases. Vaccinia virus topoisomerase I shows a definite preference for removal of positive supercoils. In contrast, topoisomerase I from human, wheat germ and Saccharomyces cerevisiae has little preference between positive and negative supercoils. The vaccinia enzyme may have evolved for functions required for optimal viral growth. topoisomerases.

DNA Topoisomerases, Type I↗
PubMed1996

Effect of the deletion of the sigma 32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance.

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a delta 32-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is > 10-fold more sensitive to heat treatment (52 degrees C) than the wild type. After brief treatment at 42 degrees C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52 degrees C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42 C (displayed on SDS-polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.

Bacterial Proteins↗