Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting.

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited 'closed' state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active 'open' state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants (i.e. poorly processive, slow, and mechanochemically uncoupled) can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower RNA binding kinetics and enhanced ATP-stimulated RNA dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2 and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

Heterogeneous distribution of kinesin-streptavidin complexes revealed by Mass Photometry.

Kinesin-streptavidin complexes are widely used in microtubule-based active-matter studies. The stoichiometry of the complexes is empirically tuned but experimentally challenging to determine. Here, mass photometry measurements reveal heterogenous distributions of kinesin-streptavidin complexes. Our binding model indicates that heterogeneity arises from both the kinesin-streptavidin mixing ratio and the kinesin-biotinylation efficiency.

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

Dual-Emissive Mn-Doped Lead Halide Perovskite Nanocrystals as Background-Suppressed Latent Fingerprint Detection Probes.

Diverse strategies have been developed to visualize latent fingerprints (LFPs) that are undetectable by the naked eye. Among them, fluorescence-based approaches have emerged as an attractive method for enabling high-resolution LFP imaging. However, the use of fluorescent probes for LFP detection remains challenging due to cumbersome processing, low selectivity, and high background interference. Here, we demonstrate highly efficient, sensitive, and background-free LFP detection with dual-color emission arising from manganese (Mn)-doped lead halide perovskite (CsPb(Cl1-yBry)3) nanocrystals (NCs). The resulting bright, fluorescent, solid-state nanopowder (NP) permits the visualization of LFP ridge structures and the resolution of level 1-3 LFP features. The dual-color emission of the Mn-doped perovskite NP provides a simple, robust, and effective route to overcome background interference, thereby increasing the resolution and sensitivity of the LFP detection. The combination of the high quantum efficiency and dual emission of Mn-doped perovskite NP offers great potential for forensic science.

Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy.

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility.

Nonlinear effects in optical trapping of titanium dioxide and diamond nanoparticles.

Optical trapping in biophysics typically uses micron-scale beads made of materials like polystyrene or glass to probe the target of interest. Using smaller beads made of higher-index materials could increase the time resolution of these measurements. We characterized the trapping of nanoscale beads made of diamond and titanium dioxide (TiO2) in a single-beam gradient trap. Calculating theoretical expectations for the trapping stiffness of these beads, we found good agreement with measured values. Trap stiffness was significantly higher for TiO2 beads, owing to notable enhancement from nonlinear optical effects, not previously observed for continuous-wave trapping. Trap stiffness was over 6-fold higher for TiO2 beads than polystyrene beads of similar size at 70 mW laser power. These results suggest that diamond and TiO2 nanobeads can be used to improve time resolution in optical tweezers measurements.

Mesoporous Polydopamine-Encapsulated Fluorescent Nanodiamonds: A Versatile Platform for Biomedical Applications.

Fluorescent nanodiamonds (FNDs) are versatile nanomaterials with promising properties. However, efficient functionalization of FNDs for biomedical applications remains challenging. In this study, we demonstrate mesoporous polydopamine (mPDA) encapsulation of FNDs. The mPDA shell is generated by sequential formation of micelles via self-assembly of Pluronic F127 (F127) with 1,3,5-trimethyl benzene (TMB) and composite micelles via oxidation and self-polymerization of dopamine hydrochloride (DA). The surface of the mPDA shell can be readily functionalized with thiol-terminated methoxy polyethylene glycol (mPEG-SH), hyperbranched polyglycerol (HPG), and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS). The PEGylated FND@mPDA particles are efficiently taken up by, and employed as a fluorescent imaging probe for, HeLa cells. HPG-functionalized FND@mPDA is conjugated with an amino-terminated oligonucleotide to detect microRNA via hybridization. Finally, the increased surface area of the mPDA shell permits efficient loading of doxorubicin hydrochloride. Further modification with TPGS increases drug delivery efficiency, resulting in high toxicity to cancer cells.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIß and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIß, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport.

Intracellular cargos are often membrane-enclosed and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Our results demonstrate that attaching kinesin to a fluid lipid membrane reduces the inhibitory effect of tau on kinesin. Moreover, adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin binding in a dosage-dependent manner. We propose that reduction of kinesin diffusion in the cargo membrane underlies the effect of cholesterol on kinesin binding in the presence of tau, and we provide a simple model for this proposed mechanism. Our study establishes a direct link between cargo membrane cholesterol and MAP-based regulation of kinesin-1. The cholesterol effects uncovered here may more broadly extend to other lipid alterations that impact motor diffusion in the cargo membrane, including those associated with aging and neurological diseases.

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Recognition of DNA Supercoil Handedness during Catenation Catalyzed by Type II Topoisomerases.

Although the presence of catenanes (i.e., intermolecular tangles) in chromosomal DNA stabilizes interactions between daughter chromosomes, a lack of resolution can have serious consequences for genomic stability. In all species, from bacteria to humans, type II topoisomerases are the enzymes primarily responsible for catenating/decatenating DNA. DNA topology has a profound influence on the rate at which these enzymes alter the superhelical state of the double helix. Therefore, the effect of supercoil handedness on the ability of human topoisomerase IIα and topoisomerase IIß and bacterial topoisomerase IV to catenate DNA was examined. Topoisomerase IIα preferentially catenated negatively supercoiled over positively supercoiled substrates. This is opposite to its preference for relaxing (i.e., removing supercoils from) DNA and may prevent the enzyme from tangling the double helix ahead of replication forks and transcription complexes. The ability of topoisomerase IIα to recognize DNA supercoil handedness during catenation resides in its C-terminal domain. In contrast to topoisomerase IIα, topoisomerase IIß displayed little ability to distinguish DNA geometry during catenation. Topoisomerase IV from three bacterial species preferentially catenated positively supercoiled substrates. This may not be an issue, as these enzymes work primarily behind replication forks. Finally, topoisomerase IIα and topoisomerase IV maintain lower levels of covalent enzyme-cleaved DNA intermediates with catenated over monomeric DNA. This allows these enzymes to perform their cellular functions in a safer manner, as catenated daughter chromosomes may be subject to stress generated by the mitotic spindle that could lead to irreversible DNA cleavage.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an 'active helicase,' capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3ß.

In metazoans, topoisomerase 3ß (TOP3B) regulates R-loop dynamics and mRNA translation, which are critical for genome stability, neurodevelopment and normal aging. As a Type IA topoisomerase, TOP3B acts by general acid-base catalysis to break and rejoin single-stranded DNA. Passage of a second DNA strand through the transient break permits dissipation of hypernegative DNA supercoiling and catenation/knotting. Additionally, hsTOP3B was recently demonstrated as the human RNA topoisomerase, required for normal neurodevelopment and proposed to be a potential anti-viral target upon RNA virus infection. Here we elucidate the biochemical mechanisms of human TOP3B. We delineate the roles of divalent metal ions, and of a conserved Lysine residue (K10) in the differential catalysis of DNA and RNA. We also demonstrate that three regulatory factors fine-tune the catalytic performance of TOP3B: the TOP3B C-terminal tail, its protein partner TDRD3, and the sequence of its DNA/RNA substrates.

The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom's syndrome helicase for efficient disruption of D-loops.

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom's syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM's multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM's interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.

General Method to Increase Carboxylic Acid Content on Nanodiamonds.

Carboxylic acid is a commonly utilized functional group for covalent surface conjugation of carbon nanoparticles that is typically generated by acid oxidation. However, acid oxidation generates additional oxygen containing groups, including epoxides, ketones, aldehydes, lactones, and alcohols. We present a method to specifically enrich the carboxylic acid content on fluorescent nanodiamond (FND) surfaces. Lithium aluminum hydride is used to reduce oxygen containing surface groups to alcohols. The alcohols are then converted to carboxylic acids through a rhodium (II) acetate catalyzed carbene insertion reaction with tert-butyl diazoacetate and subsequent ester cleavage with trifluoroacetic acid. This carboxylic acid enrichment process significantly enhanced nanodiamond homogeneity and improved the efficiency of functionalizing the FND surface. Biotin functionalized fluorescent nanodiamonds were demonstrated to be robust and stable single-molecule fluorescence and optical trapping probes.

Topoisomerase VI is a chirally-selective, preferential DNA decatenase.

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase; however, robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.

Highly stable cesium lead bromide perovskite nanocrystals for ultra-sensitive and selective latent fingerprint detection.

Latent fingerprints (LFPs) are one of the most important forms of evidence in crime scenes due to the uniqueness and permanence of the friction ridges in fingerprints. Therefore, an efficient method to detect LFPs is crucial in forensic science. However, there remain several challenges with traditional detection strategies including low sensitivity, low contrast, high background, and complicated processing steps. In order to overcome these drawbacks, we present an approach for developing latent fingerprints using stabilized CsPbBr3 perovskite nanocrystals (NCs) as solid-state nanopowders. We demonstrate the superior optical stability of CsPbBr3 NCs with respect to absorption, photoluminescence (PL), and fluorescence lifetime. We then used these highly stable, fluorescent CsPbBr3 NCs as a powder dusting material to develop LFPs on diverse surfaces. The stable optical properties and hydrophobic surface of the CsPbBr3 NC nanopowder permitted high resolution images from which unique features of friction ridge arrangements with first, second, and third-level LFP details can be obtained within minutes.

CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning.

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.