Selective inhibition of bacterial topoisomerase I

The present invention provides novel bisbenzimidazole compounds and methods of using the compounds as antibacterial agents

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein– nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins

Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases

DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (−) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (−) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (−) supercoils enhance LacI’s DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions

Biochemical and biophysical properties of positively supercoiled DNA

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (-) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (-) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (-) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (-) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (-) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (-) sc DNA

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes ( and DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, , , and . We found that for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5\u27-triphosphate binding to DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors

HMGN1 Modulates Nucleosome Occupancy and DNase I Hypersensitivity at the CpG Island Promoters of Embryonic Stem Cells

Chromatin structure plays a key role in regulating gene expression and embryonic differentiation; however, the factors that determine the organization of chromatin around regulatory sites are not fully known. Here we show that HMGN1, a nucleosome-binding protein ubiquitously expressed in vertebrate cells, preferentially binds to CpG island-containing promoters and affects the organization of nucleosomes, DNase I hypersensitivity, and the transcriptional profile of mouse embryonic stem cells and neural progenitors. Loss of HMGN1 alters the organization of an unstable nucleosome at transcription start sites, reduces the number of DNase I-hypersensitive sites genome wide, and decreases the number of nestin-positive neural progenitors in the subventricular zone (SVZ) region of mouse brain. Thus, architectural chromatin-binding proteins affect the transcription profile and chromatin structure during embryonic stem cell differentiation

DNA linking number change induced by sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins play a key role in many fundamental biological processes, such as transcription, DNA replication and recombination. Very often, these DNA-binding proteins introduce structural changes to the target DNA-binding sites including DNA bending, twisting or untwisting and wrapping, which in many cases induce a linking number change (ΔLk) to the DNA-binding site. Due to the lack of a feasible approach, ΔLk induced by sequence-specific DNA-binding proteins has not been fully explored. In this paper we successfully constructed a series of DNA plasmids that carry many tandem copies of a DNA-binding site for one sequence-specific DNA-binding protein, such as λ O, LacI, GalR, CRP and AraC. In this case, the protein-induced ΔLk was greatly amplified and can be measured experimentally. Indeed, not only were we able to simultaneously determine the protein-induced ΔLk and the DNA-binding constant for λ O and GalR, but also we demonstrated that the protein-induced ΔLk is an intrinsic property for these sequence-specific DNA-binding proteins. Our results also showed that protein-mediated DNA looping by AraC and LacI can induce a ΔLk to the plasmid DNA templates. Furthermore, we demonstrated that the protein-induced ΔLk does not correlate with the protein-induced DNA bending by the DNA-binding proteins

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals