Biology subjects

Tse-Dinh, Y.-C.

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

10 recordsLinked to original sources

bioRxiv2026

Coordinated topoisomerase function shapes the fluoroquinolone response of Chlamydia trachomatis

DNA supercoiling is essential for the developmental cycle of Chlamydia trachomatis, yet its role in shaping antibiotic responses remains poorly understood. We investigated how the fluoroquinolone moxifloxacin (Mox) influenced C. trachomatis growth across developmental stages with its distinct supercoiling levels. Early Mox exposure completely halted bacterial growth, whereas treatment during mid-cycle produced enlarged, persistent forms and abolished formation of infectious progeny. These stage-specific outcomes coincided with inhibition of DNA replication, depletion of DNA gyrase, and transcriptional repression of ompA and omcB, accompanied by preserved or elevated expression of the stress-responsive groESL1 operon. Mox also elicited compensatory downregulation of topoisomerase I (TopA), consistent with attempts to rebalance intracellular supercoiling. Together, these data demonstrate that fluoroquinolone susceptibility in C. trachomatis reflects stage-dependent supercoiling levels. Perturbation of supercoiling homeostasis drives developmental arrest and persistence phenotypes, highlighting coordinated gyrase-TopA activity as a key determinant of fluoroquinolone tolerance and a potential target for overcoming persistent infection. ImportanceC. trachomatis, a medically significant bacterial pathogen, can persist under antimicrobial pressure, complicating treatment strategy. This study links supercoiling homeostasis to fluoroquinolone tolerance, offering mechanistic insights into chlamydial adaptation to antibiotic stress and identifying potential targets to overcome persistence--an urgent challenge in global reproductive health.

microbiology↗

bioRxiv2026

Investigation of potential hinge region for Mycobacterium tuberculosis topoisomerase I conformational change during catalysis

Mycobacterium tuberculosis topoisomerase I (MtbTOP1) is essential for the viability of the causative agent of TB. There are still significant unanswered questions regarding the dynamic conformations during catalysis of relaxation of negatively supercoiled DNA by MtbTOP1. We aim to study the flexible hinge residues that control the dynamics of inter-domain rearrangements involved in the enzyme conformational changes that allow the opening-closing of the topoisomerase gate. We used the online server PACKMAN to predict possible hinges from the MtbTOP1 crystal structure. The predicted region "PRO506 to LEU526" at the border between domains D2 and D4 with a p-value <0.05 was then studied as a potential hinge. The highly conserved ARG516 from this region interacts with the DNA inside the protein toroidal cavity. This arginine maintains inter-domain interaction with GLU207 of D4 and ASP691 of D5 domains. After introducing alanine substitutions, we further studied the mutant topoisomerases in biochemical experiments. The results showed a significant loss in DNA relaxation activity without affecting DNA binding and cleavage after mutating GLU207 and ARG516, consistent with their role as hinge residues in domain rearrangements.

biochemistry↗

bioRxiv2025

Identification of novel human topoisomerase III beta inhibitors

Human topoisomerase III beta (TOP3B) is a type IA topoisomerase that can change the topology of DNA and RNA substrates via a phosphotyrosine covalent intermediate. TOP3B has been shown to be required for the efficient replication of certain positive-sense ssRNA viruses including Dengue. We applied molecular dynamics simulation combined with docking studies to identify potential inhibitors of TOP3B from a library comprised of drugs that are FDA-approved or undergoing clinical trials for potential drug repurposing. Topoisomerase activity assay of the top virtual screening hits showed that bemcentinib, a compound known to target the AXL receptor tyrosine kinase, can inhibit TOP3B relaxation activity. Additional small molecules that share the N5,N3-1H-1,2,4-triazole-3,5-diamine moiety of bemcentinib were synthesized and tested for inhibition of TOP3B relaxation activity. Five of these molecules showed comparable IC50 as bemcentinib for inhibition of TOP3B. However, these five molecules had less selectivity towards TOP3B inhibition versus bemcentinib when inhibition of the type IB human topoisomerase I was com-pared. These results suggest that exploration of tyrosine kinase inhibitors and their analogs may allow the identification of novel topoisomerase inhibitors.

biochemistry↗

bioRxiv2024

The SWIB domain-containing DNA topoisomerase I of Chlamydia trachomatis mediates DNA relaxation

The obligate intracellular bacterial pathogen, Chlamydia trachomatis (Ct), has a distinct DNA topoisomerase I (TopA) with a C-terminal domain (CTD) homologous to eukaryotic SWIB domains. Despite the lack of sequence similarity at the CTDs between C. trachomatis TopA (CtTopA) and Escherichia coli TopA (EcTopA), full-length CtTopA removed negative DNA supercoils in vitro and complemented the growth defect of an E. coli topA mutant. We demonstrated that CtTopA is less processive in DNA relaxation than EcTopA in dose-response and time course studies. An antibody generated against the SWIB domain of CtTopA specifically recognized CtTopA but not EcTopA or Mycobacterium tuberculosis TopA (MtTopA), consistent with the sequence differences in their CTDs. The endogenous CtTopA protein is expressed at a relatively high level during the middle and late developmental stages of C. trachomatis. Conditional knockdown of topA expression using CRISPRi in C. trachomatis resulted in not only a developmental defect but also in the downregulation of genes linked to nucleotide acquisition from the host cells. Because SWIB-containing proteins are not found in prokaryotes beyond Chlamydia spp., these results imply a significant function for the SWIB-containing CtTopA in facilitating the energy metabolism of C. trachomatis for its unique intracellular growth. ImportanceC. trachomatis (Ct) is a medically important bacterial pathogen that is responsible for the most prevalent sexually transmitted bacterial infection. Bioinformatics, genetics, and biochemical analyses have established that the presence of a SWIB domain in CtTopA, a DNA topoisomerase I, is relevant to chlamydial physiology. Further defining the mechanisms of the C-terminal SWIB domain on the catalytic function of CtTopA in an intracellular pathogen is warranted for a more complete understanding of the interactions between C. trachomatis and its host cells.

molecular biology↗

bioRxiv2024

Mycobacterial Topoisomerase IA Energetically Suffers From C-terminal Deletions

Type IA topoisomerases relieve torsional stress in DNA by a strand-passage mechanism, using the strain in the DNA to drive relaxation. The topoisomerase IAs of the Mycobacterium genus have distinct C-terminal domains which are crucial for successful strand-passage. We used single-molecule magnetic tweezers to observe supercoil relaxation by wild type Mycobacterium smegmatis topoisomerase IA and two C-terminal truncation mutants. We recorded distinct behaviors from each truncation mutant. We calculated the free energy stored in the DNA as it is twisted under force to examine the differences between the proteins. Based on our results, we propose a modified model of the strand-passage cycle.

biophysics↗

bioRxiv2021

A Yeast-Based Screening System for Differential Identification of Poison Inhibitors and Catalytic Inhibitors of Human Topoisomerase I

1.Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern) - induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1 covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poison inhibitors developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. The aim of this study is to develop a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. This yeast-based screening system can identify human TOP1 poison inhibitors for anticancer efficacy as well as catalytic inhibitors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. In addition to distinguishing between such TOP1 catalytic inhibitors and TOP1 poison inhibitors, results from this yeast-based screening system will also allow elimination of compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

molecular biology↗

bioRxiv2020

Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

Genetic inactivation of TOP3B is linked with schizophrenia, autism, intellectual disability and cancer. The present study demonstrates that in vivo TOP3B forms both RNA and DNA cleavage complexes (TOP3Bccs) and reveals a pathway for repairing TOP3Bccs. For detecting cellular TOP3Bccs, we engineered a "self-trapping" mutant of TOP3B (R338W TOP3B) and to determine how human cells repair TOP3Bccs, we depleted tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells produced elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowered cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 cannot excise the native form of TOP3Bccs. Hypothesizing that TDP2 cannot access phosphotyrosyl linkage unless TOP3B is either proteolyzed or denatured, we found that cellular TOP3Bccs are ubiquitinated by the E3 Ubiquitin Ligase TRIM41 before undergoing proteasomal degradation and excision by TDP2. HIGHLIGHTSO_LIMethod for in vivo detection of TOP3B cleavage complexes (TOP3Bccs) formed both in DNA and RNA, using a religation defective "self-trapping" R338W TOP3B mutant. C_LIO_LIFirst evidence that TDP2 excises TOPccs produced by a type IA topoisomerase. C_LIO_LITDP2 processes both RNA and DNA TOP3Bccs following their ubiquitylation and proteasomal degradation inside cell. C_LIO_LITRIM41 is the first reported E3 ubiquitin ligase for TOP3Bcc ubiquitylation and proteasomal degradation. C_LI

cell biology↗

bioRxiv2019

Tyrosyl-DNA phosphodiesterase 1 and topoisomerase I activities as predictive indicators for Glioblastoma susceptibility to genotoxic agents

BackgroundGlioblastoma (GBM) patients have an estimated survival of [~]15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan, currently in clinical trials for GBM, targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc) inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan.\n\nMethodsWe treated GBM cell lines with increasing concentrations of irinotecan and compared the IC50 values. TOP1 and TDP1 protein levels from each cell type as well as GBM patient tumors were determined by Western blot analysis, while activity levels were ascertained by specific enzymatic assays. Cellular TDP1 was elevated by ectopic expression of wild-type or mutant TDP1.\n\nResultsAfter comparing cellular susceptibility to TDP1 and TOP1 concentrations and activities, we found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.92) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by ectopic expression of wild-type TDP1 increased in irinotecan IC50, while expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan.\n\nConclusionsTDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies.\n\nKey PointsO_LITDP1/TOP1 activity ratio correlates with irinotecan sensitivity in GBM cell lines.\nC_LIO_LITDP1 and TOP1 protein levels are not reliable predictors for irinotecan activity.\nC_LIO_LITDP1 inhibition is a plausible approach to improve irinotecan effectiveness in GBM.\nC_LI\n\nImportance of the StudyThe current standard of care (surgery, radiation, and chemotherapy) for GBM patients modestly enhances survival beyond [~]15 months. Thus, there is a great need for effective therapies and biomarkers that address personalized variations in GBM tumors to improve treatment outcome. Topoisomerase I (TOP1) is the target of irinotecan. The repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) is known to excise irinotecan-induced TOP1-DNA cleavage complexes (TOP1ccs). Consequently, this study examines the relationship between TOP1 and TDP1 expression and activities in GBM cells and their correlation with irinotecan sensitivity. The results reveal that the TDP1/TOP1 activity ratio predicts irinotecan vulnerability in GBM cell lines. TDP1/TOP1 activity ratio was found to vary among GBM patient tumors. This potential predictive indicator may permit selection of patients responsive to irinotecan based on the capacity to repair TOP1cc. Moreover, inhibitors of TDP1 may represent a promising approach to enhance irinotecan efficacy in GBM patients.

cancer biology↗

bioRxiv2018

Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 M against Mycobacterium tuberculosis, SI = 9.8). Here, we found that the compound is bactericidal towards Mycobacterium smegmatis, resulting in greater than 5 Log10 reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 M (4X MIC) concentration. Growth inhibition (MIC = 50 M) and reduction in cfu could also be observed against a clinical isolate of Mycobacterium abscessus. Stepwise isolation of resistant mutants of M. smegmatis was conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibits Mtb topoisomerase I and Mtb gyrase with IC50 of 0.24 and 31.5 M, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC50 for inhibition of Mtb topoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of Mtb topoisomerase I.

microbiology↗

bioRxiv2018

Direct Observation of Topoisomerase IA Gate Dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

biophysics↗