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Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

ABSTRACT: Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.

SUBMITTER: Ba S

PROVIDER: S-EPMC8650243 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

Ba Sai S Gao Guangpeng G Li Tianhu T Zhang Hao H

Journal of nanobiotechnology 20211207 1

Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expres ...[more]

PMID: 34876137

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Project description:This study catalogs production of industrially important enzymes and changes in transcript expression caused by 2-deoxy D-glucose (2-DG) treatment in Arthrinium malaysianum cultures. Carbon Catabolite Repression (CCR) induced by 2-DG in this species is cAMP independent unlike many other organisms. Higher levels of secreted endoglucanase (EG), β-glucosidase (BGL), β-xylosidase (BXL), and filter paper activity assay (FPase) enzymes under 2-DG treatment can be exploited for commercial purposes. An integrated RNA sequencing and quantitative proteomic analysis was performed to investigate the cellular response to 2-DG in A. malaysianum. Analysis of RNASeq data under 2-DG treated and control condition reveals that 56% of the unigenes do not have any known similarity to proteins in non-redundant database. Gene Ontology IDs were assigned to 36% of the transcripts (13260) and about 5207 (14%) were mapped to Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). About 1711 genes encoding 2691 transcripts were differentially expressed in treated vs. control samples. Out of the 2691 differentially expressed transcripts, only 582 have any known function. The most up regulated genes belonged to Pentose Phosphate Pathways and carbohydrate degradation class as expected. In addition, genes involved in protein folding, binding, catalytic activity, DNA repair, and secondary metabolites were up-regulated under 2-DG treatment. Whereas genes encoding glycosylation pathways, growth, nutrient reservoir activity was repressed. Gene ontology analysis of the differentially expressed genes indicates metabolic process (35%) is the pre-dominant class followed by carbohydrate degradation (11%), protein folding, and trafficking (6.2%) and transport (5.3%) classes. Unlike other organisms, conventional unfolded protein response (UPR) was not activated in either control or treated conditions. Major enzymes secreted by A. malaysianum are those degrading plant polysaccharides, the most dominant ones being β-glucosidase, as demonstrated by the 2D gel analysis. A set of 7 differentially expressed mRNAs were validated by qPCR. Transmission electron microscopy analyses demonstrated that the 2-DG treated cell walls of hyphae showed significant differences in the cell-wall thickness. Overall 2-DG treatment in A. malaysianum induced secretion of large amount of commercially viable enzymes compared to other known species.

Project description:The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.

Dataset Information

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

Publications

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

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