142 results on '"Keir C. Neuman"'
Search Results
2. The toposiomerase IIIalpha-RMI1-RMI2 complex orients human Bloom’s syndrome helicase for efficient disruption of D-loops
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Gábor M. Harami, János Pálinkás, Yeonee Seol, Zoltán J. Kovács, Máté Gyimesi, Hajnalka Harami-Papp, Keir C. Neuman, and Mihály Kovács
- Subjects
Science - Abstract
Human Bloom’s syndrome (BLM) helicase has a role in DNA repair, and BLM deficiency in humans is associated with chromosomal abnormalities. Here the authors employ solution biophysical assays to show BLM maintains a balance for disruption and stabilization of oligonucleotide-based D-loops. Interaction with the Topoisomerase IIIalpha-RMI1-RMI2 complex orients the activity toward D-loop disruption.
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- 2022
- Full Text
- View/download PDF
3. General Method to Increase Carboxylic Acid Content on Nanodiamonds
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Ganesh Shenoy, Jessica Ettedgui, Chandrasekhar Mushti, Jennifer Hong, Kelly Lane, Burchelle Blackman, Hak-Sung Jung, Yasuharu Takagi, Yeonee Seol, Martin Brechbiel, Rolf E. Swenson, and Keir C. Neuman
- Subjects
fluorescent nanodiamond ,optical trapping ,TIRF ,rhodium catalysis ,functionalization ,Organic chemistry ,QD241-441 - Abstract
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.
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- 2022
- Full Text
- View/download PDF
4. Distribution bias and biochemical characterization of TOP1MT single nucleotide variants
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Hongliang Zhang, Yeonee Seol, Keli Agama, Keir C. Neuman, and Yves Pommier
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Medicine ,Science - Abstract
Abstract Mitochondrial topoisomerase I (TOP1MT) is a type IB topoisomerase encoded in the nucleus of vertebrate cells. In contrast to the other five human topoisomerases, TOP1MT possesses two high frequency single nucleotide variants (SNVs), rs11544484 (V256I, Minor Allele Frequency = 0.27) and rs2293925 (R525W, MAF = 0.45), which tend to be mutually exclusive across different human ethnic groups and even more clearly in a cohort of 129 US patients with breast cancer and in the NCI-60 cancer cell lines. We expressed these two TOP1MT variants and the double-variant (V256I-R525W) as recombinant proteins, as well as a less common variant E168G (rs200673353, MAF = 0.001), and studied their biochemical properties by magnetic tweezers-based supercoil relaxation and classical DNA relaxation assays. Variants showed reduced DNA relaxation activities, especially the V256I variant towards positively supercoiled DNA. We also found that the V256I variant was enriched to MAF = 0.64 in NCI-60 lung carcinoma cell lines, whereas the TOP1MT R525W was enriched to MAF = 0.65 in the NCI-60 melanoma cell lines. Moreover, TOP1MT expression correlated with the 256 variants in the NCI-60 lung carcinoma cell lines, valine with high expression and isoleucine with low expression. Our results are discussed in the context of evolution between the nuclear and mitochondrial topoisomerases and potential cancer predisposition.
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- 2017
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- View/download PDF
5. Surface Modification of Fluorescent Nanodiamonds for Biological Applications
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Hak-Sung Jung and Keir C. Neuman
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fluorescent nanodiamond ,nitrogen vacancy center ,detonation nanodiamond ,biocompatibility ,functionalization ,biological applications ,Chemistry ,QD1-999 - Abstract
Fluorescent nanodiamonds (FNDs) are a new class of carbon nanomaterials that offer great promise for biological applications such as cell labeling, imaging, and sensing due to their exceptional optical properties and biocompatibility. Implementation of these applications requires reliable and precise surface functionalization. Although diamonds are generally considered inert, they typically possess diverse surface groups that permit a range of different functionalization strategies. This review provides an overview of nanodiamond surface functionalization methods including homogeneous surface termination approaches (hydrogenation, halogenation, amination, oxidation, and reduction), in addition to covalent and non-covalent surface modification with different functional moieties. Furthermore, the subsequent coupling of biomolecules onto functionalized nanodiamonds is reviewed. Finally, biomedical applications of nanodiamonds are discussed in the context of functionalization.
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- 2021
- Full Text
- View/download PDF
6. Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques
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Shannon J. McKie, Anthony Maxwell, and Keir C. Neuman
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dna topoisomerase ,next-generation sequencing ,genome wide ,topoisomerase cleavage ,topoisomerase binding ,antibiotics ,anticancer drugs ,Genetics ,QH426-470 - Abstract
Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein−DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.
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- 2020
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7. Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy
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Daniël P Melters, Keir C Neuman, Reda S Bentahar, Tatini Rakshit, and Yamini Dalal
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epigenetics ,nucleosomes ,chromatin ,single-molecule ,high-speed AFM ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
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.
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- 2023
- Full Text
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8. Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion
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Jonathan M Craig, Maria Mills, Hwanhee C Kim, Jesse R Huang, Sarah J Abell, Jonathan W Mount, Jens H Gundlach, Keir C Neuman, and Andrew H Laszlo
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Nanopores ,Adenosine Triphosphate ,RecQ Helicases ,Nucleic Acids ,Escherichia coli ,Genetics ,DNA, Single-Stranded - Abstract
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.
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- 2022
9. Recognition of DNA Supercoil Handedness during Catenation Catalyzed by Type II Topoisomerases
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Esha D. Dalvie, Jordan C. Stacy, Keir C. Neuman, and Neil Osheroff
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DNA Topoisomerase IV ,DNA Topoisomerases, Type II ,DNA Topoisomerases, Type I ,DNA, Superhelical ,Catenanes ,Humans ,Biochemistry ,Article ,Catalysis ,Functional Laterality - Abstract
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 IIβ 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 IIβ 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.
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- 2022
10. Data from Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors
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Keir C. Neuman, Yves Pommier, Nicholas Lorence, Keli Agama, Hongliang Zhang, and Yeonee Seol
- Abstract
Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anticancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors. Mol Cancer Ther; 14(11); 2552–9. ©2015 AACR.
- Published
- 2023
11. Supplemental Figure 1-3 from Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors
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Keir C. Neuman, Yves Pommier, Nicholas Lorence, Keli Agama, Hongliang Zhang, and Yeonee Seol
- Abstract
Supplemental information contains three figures. Figure s1 displays plots of in vitro inhibition plotted versus IC50 values for inhibitors as measured for three individual cell lines. Figure S2 displays the low rate probability and religation inhibition measures of inhibition plotted versus the average normalized IC50 values for each of the four inhibitors. Figure S3 displays the fraction of relaxed DNA measured in the ensembled supercoil relaxation assay as a function of the inhibitor concentration for the four inhibitors.
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- 2023
12. Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases
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Jeffrey Y Jian, Kevin D McCarty, Jo Ann W Byl, F Peter Guengerich, Keir C Neuman, and Neil Osheroff
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Nucleic Acid Enzymes ,Genetics - Abstract
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 IIβ 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/IIβ, 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.
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- 2023
13. Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport
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Qiaochu Li, James T. Ferrare, Jonathan Silver, John O. Wilson, Luis Arteaga-Castaneda, Weihong Qiu, Michael Vershinin, Stephen J. King, Keir C. Neuman, and Jing Xu
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Multidisciplinary - Abstract
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.
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- 2023
14. UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay
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Joseph H Chapman, Jonathan M Craig, Clara D Wang, Jens H Gundlach, Keir C Neuman, and J Robert Hogg
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Adenosine Triphosphatases ,RNA and RNA-protein complexes ,Trans-Activators ,DNA Helicases ,Genetics ,Humans ,RNA ,RNA, Messenger ,RNA Helicases ,Nonsense Mediated mRNA Decay - Abstract
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 (
- Published
- 2022
15. Unraveling the molecular mechanism of type II topoisomerase poisons with magnetic tweezers
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Ian L. Morgan and Keir C. Neuman
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Biophysics - Published
- 2023
16. Cholesterol in the cargo membrane reduces kinesin-1 binding to microtubules in the presence of tau
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Qiaochu Li, James T. Ferrare, Jonathan Silver, John O. Wilson, Luis Arteaga-Castaneda, Weihong Qiu, Michael Vershinin, Stephen J. King, Keir C. Neuman, and Jing Xu
- Subjects
macromolecular substances - Abstract
Intracellular cargos are often membrane-bound 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. Attaching kinesins to a fluid lipid membrane decreases the inhibitory effect of tau in comparison to membrane-free cargos. Adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin in a dosage-dependent manner. Our findings can be understood in a framework in which kinesin diffusion in the cargo membrane counteracts binding-site occlusion by tau. Our study establishes a direct link between the physical properties of cargo membrane and MAP-based regulation of kinesin-1.
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- 2022
17. Topoisomerase VI is a chirally-selective, preferential DNA decatenase
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Shannon J McKie, Parth Rakesh Desai, Yeonee Seol, Adam MB Allen, Anthony Maxwell, and Keir C Neuman
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General Immunology and Microbiology ,QH301-705.5 ,Archaeal Proteins ,Science ,General Neuroscience ,methanosarcina mazei ,Stereoisomerism ,General Medicine ,DNA, Catenated ,Single Molecule Imaging ,General Biochemistry, Genetics and Molecular Biology ,DNA topology ,DNA Topoisomerases, Type II ,topoisomerase IIB ,Methanosarcina ,Medicine ,single-molecule ,Biology (General) ,magnetic tweezers - Abstract
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°.
- Published
- 2022
18. Single Molecule Analysis of CENP-A Chromatin by High-Speed Atomic Force Microscopy
- Author
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Daniël P. Melters, Keir C. Neuman, Tatini Rakshit, and Yamini Dalal
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macromolecular substances - Abstract
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 in 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 dynamicsin vitroinform us about chromatin accessibilityin vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the MSD and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, 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 dynamicsin vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. Thus, changes which alter chromatin accessibilityin vitro, also correspondingly alter transcriptionin vivo. These data suggest a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance mobility.
- Published
- 2022
19. Biocompatible Fluorescent Nanodiamonds as Multifunctional Optical Probes for Latent Fingerprint Detection
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Keir C. Neuman, Seungjin Ryu, Kyung-Jin Cho, Hak-Sung Jung, Paul A. Roche, and Yasuharu Takagi
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Materials science ,genetic structures ,Biocompatibility ,technology, industry, and agriculture ,Nanoparticle ,macromolecular substances ,02 engineering and technology ,Adhesion ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Fluorescence ,Article ,Latent fingerprint ,0104 chemical sciences ,Nanomaterials ,Cell killing ,Biophysics ,General Materials Science ,0210 nano-technology ,Binding selectivity - Abstract
There is an immense literature on detection of latent fingerprints (LFPs) with fluorescent nanomaterials because fluorescence is one of the most sensitive detection methods. Although many fluorescent probes have been developed for latent fingerprint detection, many challenges remain, including the low selectivity, complicated processing, high background, and toxicity of nanoparticles used to visualize LFPs. In this study, we demonstrate biocompatible, efficient, and low background LFP detection with poly(vinylpyrrolidone) (PVP) coated fluorescent nanodiamonds (FNDs). PVP-coated FND (FND@PVP) is biocompatible at the cellular level. They neither inhibit cellar proliferation nor induce cell death via apoptosis or other cell killing pathways. Moreover, they do not elicit an immune response in cells. PVP coating enhances the physical adhesion of FND to diverse substrates and in particular results in efficient binding of FND@PVP to fingerprint ridges due to the intrinsic amphiphilicity of PVP. Clear, well-defined ridge structures with first, second, and third-level of LFP details are revealed within minutes by FND@PVP. The combination of this binding specificity and the remarkable optical properties of FND@PVP permits the detection of LPFs with high contrast, efficiency, selectivity, sensitivity, and reduced background interference. Our results demonstrate that background-free imaging via multicolor emission and dual-modal imaging of FND@PVP nanoparticles have great potential for high-resolution imaging of LFPs.
- Published
- 2020
20. Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion
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Jesse R. Huang, Hwanhee C. Kim, Maria Mills, Jonathan M. Craig, Jonathan W. Mount, Andrew H. Laszlo, Keir C. Neuman, Sarah J. Abell, and Jens H. Gundlach
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Physics ,biology ,RecQ Helicases ,Helicase ,Energy landscape ,Coupling (electronics) ,Nanopore ,chemistry.chemical_compound ,chemistry ,Tweezers ,biology.protein ,Biophysics ,Spatiotemporal resolution ,DNA - Abstract
Helicases are essential for nearly all nucleic acid processes across the tree of life. Using Nanopore Tweezers we observed the small, fast steps taken by single RecQ helicases as they step along and unwind DNA at ultrahigh spatiotemporal resolution. By directly measuring conformational substates of RecQ we determine the coupling between helicase domain motions and chemical reactions that together produce forward motion along the DNA. Application of assisting and opposing forces shows that RecQ has a highly asymmetric energy landscape that reduces its sensitivity to opposing mechanical forces that could be encountered in vivo by molecular roadblocks such as DNA bound proteins. This energy landscape enables RecQ to maintain speed against an opposing load.
- Published
- 2021
21. Author response: CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning
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James A. Taylor, Kiyoshi Mizuuchi, Yeonee Seol, Jagat B. Budhathoki, and Keir C. Neuman
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Complex dynamics ,chemistry.chemical_compound ,biology ,Chemistry ,ATPase ,Partition (politics) ,biology.protein ,Biophysics ,DNA - Published
- 2021
22. CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning
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Jagat B. Budhathoki, James A. Taylor, Keir C. Neuman, Kiyoshi Mizuuchi, and Yeonee Seol
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DNA, Bacterial ,Magnetic tweezers ,QH301-705.5 ,ATPase ,Science ,Cytidine Triphosphate ,Centromere ,chromosome segregation ,plasmid partition ,DNA Primase ,General Biochemistry, Genetics and Molecular Biology ,Chromosome segregation ,chemistry.chemical_compound ,Plasmid ,Bacterial Proteins ,ATP hydrolysis ,Escherichia coli ,Partition (number theory) ,Biology (General) ,Pyrophosphatases ,diffusion-ratchet ,Adenosine Triphosphatases ,Microbiology and Infectious Disease ,General Immunology and Microbiology ,biology ,Base Sequence ,General Neuroscience ,Circular bacterial chromosome ,Escherichia coli Proteins ,E. coli ,General Medicine ,Cell Biology ,Chromosomes, Bacterial ,CTPase ,DNA-Binding Proteins ,chemistry ,ParB spreading ,biology.protein ,Biophysics ,Medicine ,magnetic tweezers ,DNA ,Research Article ,Plasmids ,Protein Binding - Abstract
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.
- Published
- 2021
23. How UPF1 helicase activity facilitates mRNA decay
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Joseph H. Chapman, Keir C. Neuman, and J.R. Hogg
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Biophysics - Published
- 2022
24. Glutamate Brings Out the Flavor of SSB Cooperativity and Phase Separation
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Gabor M. Harami and Keir C. Neuman
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Structural Biology ,Molecular Biology - Published
- 2022
25. Direct Observation of Topoisomerase IA Gate Dynamics
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Yuk-Ching Tse-Dinh, Maria Mills, and Keir C. Neuman
- Subjects
0301 basic medicine ,DNA, Bacterial ,Models, Molecular ,Magnetic tweezers ,DNA, Single-Stranded ,Cleavage (embryo) ,Article ,03 medical and health sciences ,Molecular dynamics ,chemistry.chemical_compound ,Structural Biology ,Cleave ,Escherichia coli ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Topoisomerase ,030302 biochemistry & molecular biology ,Kinetics ,030104 developmental biology ,DNA Topoisomerases, Type I ,Biophysics ,biology.protein ,DNA supercoil ,Ligation ,AND gate ,DNA - Abstract
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 type IA topoisomerases are assumed to 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 Escherichia coli type IA topoisomerases I and III. We found that after 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 allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA. Single-molecule magnetic tweezers analyses and supporting MD simulations provide evidence for protein-gate opening of type IA topoisomerases.
- Published
- 2018
26. Topoisomerase VI is a chirally-selective, preferential DNA decatenase
- Author
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Parth Rakesh Desai, Anthony Maxwell, Keir C. Neuman, Yeonee Seol, and Shannon J. Mckie
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Magnetic tweezers ,biology ,Chemistry ,Topoisomerase ,biology.organism_classification ,chemistry.chemical_compound ,biology.protein ,Biophysics ,DNA supercoil ,A-DNA ,Bacteria ,DNA ,Writhe ,Archaea - Abstract
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 dramatic 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°.
- Published
- 2021
27. CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning
- Author
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James A. Taylor, Yeonee Seol, Jagat Budhathoki, Keir C. Neuman, and Kiyoshi Mizuuchi
- Subjects
Complex dynamics ,chemistry.chemical_compound ,Plasmid ,biology ,chemistry ,ATP hydrolysis ,Circular bacterial chromosome ,ATPase ,Kinetics ,biology.protein ,Biophysics ,Partition (number theory) ,DNA - Abstract
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.
- Published
- 2021
28. DNA topoisomerases: Advances in understanding of cellular roles and multi-protein complexes via structure-function analysis
- Author
-
Anthony Maxwell, Keir C. Neuman, and Shannon J. Mckie
- Subjects
0303 health sciences ,biology ,Chemistry ,Mechanism (biology) ,Topoisomerase ,Structure function ,Computational biology ,DNA ,DNA gyrase ,General Biochemistry, Genetics and Molecular Biology ,DNA metabolism ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,DNA Topoisomerases, Type II ,DNA Topoisomerases, Type I ,biology.protein ,DNA supercoil ,030217 neurology & neurosurgery ,Function (biology) ,DNA Topoisomerases ,030304 developmental biology - Abstract
DNA topoisomerases, capable of manipulating DNA topology, are ubiquitous and indispensable for cellular survival due to the numerous roles they play during DNA metabolism. As we review here, current structural approaches have revealed unprecedented insights into the complex DNA-topoisomerase interaction and strand passage mechanism, helping to advance our understanding of their activities in vivo. This has been complemented by single-molecule techniques, which have facilitated the detailed dissection of the various topoisomerase reactions. Recent work has also revealed the importance of topoisomerase interactions with accessory proteins and other DNA-associated proteins, supporting the idea that they often function as part of multi-enzyme assemblies in vivo. In addition, novel topoisomerases have been identified and explored, such as topo VIII and Mini-A. These new findings are advancing our understanding of DNA-related processes and the vital functions topos fulfil, demonstrating their indispensability in virtually every aspect of DNA metabolism.
- Published
- 2020
29. Coarse-grained modelling of DNA plectoneme pinning in the presence of base-pair mismatches
- Author
-
Keir C. Neuman, Siddhartha Das, Sumitabha Brahmachari, Parth Rakesh Desai, and John F. Marko
- Subjects
0303 health sciences ,Materials science ,Base pair ,Base Pair Mismatch ,Dynamics (mechanics) ,Computational Biology ,Bending ,DNA ,Biology ,Molecular Dynamics Simulation ,01 natural sciences ,Nucleobase ,03 medical and health sciences ,Molecular dynamics ,chemistry.chemical_compound ,chemistry ,Chemical physics ,0103 physical sciences ,Genetics ,DNA supercoil ,Coarse-grained modeling ,010306 general physics ,030304 developmental biology - Abstract
Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo , defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting mean-field theory to study the effect of mismatched base pairs on DNA supercoiling. The coarse-grained approach reproduces experimentally observed deterministic plectoneme pinning at the mismatch under conditions of relatively high force (> 2 pN) and high salt concentration (> 0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. The simulation results broadly agree with existing statistical mechanical mean-field theoretical predictions for the high force-high salt regime. The coarse-grained simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.
- Published
- 2020
30. Bimodal actions of a naphthyridone/aminopiperidine-based antibacterial that targets gyrase and topoisomerase IV
- Author
-
Keir C. Neuman, Alexandria A. Oviatt, Elizabeth G. Gibson, Pan F. Chan, Neil Osheroff, and Monica Cacho
- Subjects
DNA Topoisomerase IV ,DNA, Bacterial ,Indoles ,Topoisomerase IV ,medicine.drug_class ,Pyridones ,Antibiotics ,DNA, Single-Stranded ,Pharmacology ,Biochemistry ,DNA gyrase ,Article ,03 medical and health sciences ,Antibiotic resistance ,Piperidines ,medicine ,Escherichia coli ,Topoisomerase II Inhibitors ,DNA Breaks, Double-Stranded ,DNA Cleavage ,Naphthyridines ,0303 health sciences ,biology ,Chemistry ,030302 biochemistry & molecular biology ,Mycobacterium tuberculosis ,Anti-Bacterial Agents ,Drug class ,DNA Gyrase ,Bacillus anthracis ,biology.protein - Abstract
Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV, but interact differently with the enzymes. This has led to the development of the “novel bacterial topoisomerase inhibitor” (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases including gyrase from Mycobacterium tuberculosis, and gyrase and topoisomerase IV from Bacillus anthracis and Escherichia coli. GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.
- Published
- 2019
31. A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair
- Author
-
Sanjay Kumar Bharti, Lynda Bradley, Robert M. Brosh, Joshua A. Sommers, Keir C. Neuman, Irfan Khan, Sanket Awate, Marina A. Bellani, Kazuo Shin-ya, Graeme A. King, Koji Kobayashi, Yuliang Wu, Dana Branzei, Marc S. Wold, Takuye Abe, Yeonee Seol, Hiroyuki Kitao, and Venkatasubramanian Vidhyasagar
- Subjects
0301 basic medicine ,Aphidicolin ,DNA Replication ,DNA Repair ,DNA damage ,DNA repair ,Mutation, Missense ,DNA, Single-Stranded ,Cell Line ,Recombinases ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Stress, Physiological ,Replication Protein A ,Genetics ,Animals ,DNA Breaks, Double-Stranded ,Replication protein A ,Oxazoles ,Adenosine Triphosphatases ,biology ,Nucleic Acid Enzymes ,DNA replication ,DNA Helicases ,Helicase ,Processivity ,Fanconi Anemia Complementation Group Proteins ,Cell biology ,G-Quadruplexes ,030104 developmental biology ,Fanconi Anemia ,chemistry ,030220 oncology & carcinogenesis ,Checkpoint Kinase 1 ,biology.protein ,DNA Polymerase Inhibitor ,Rad51 Recombinase ,Cisplatin ,Chickens ,RNA Helicases - Abstract
Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj−/− cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj−/− cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.
- Published
- 2018
32. Highly stable cesium lead bromide perovskite nanocrystals for ultra-sensitive and selective latent fingerprint detection
- Author
-
Junsang Cho, Keir C. Neuman, and Hak-Sung Jung
- Subjects
Bromides ,Titanium ,Photoluminescence ,Cesium ,chemistry.chemical_element ,Oxides ,Nanotechnology ,Calcium Compounds ,Biochemistry ,Fluorescence ,Article ,Latent fingerprint ,Analytical Chemistry ,Lead ,chemistry ,Nanocrystal ,Caesium ,Nanoparticles ,Environmental Chemistry ,Absorption (electromagnetic radiation) ,Spectroscopy ,Ultra sensitive ,Perovskite (structure) - Abstract
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.
- Published
- 2021
33. RecQ helicase triggers a binding mode change in the SSB–DNA complex to efficiently initiate DNA unwinding
- Author
-
Yeonee Seol, Zoltán Kovács, Máté Gyimesi, Gábor M. Harami, Máté Martina, Mihály Kovács, Keir C. Neuman, and Maria Mills
- Subjects
DNA, Bacterial ,Models, Molecular ,0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Magnetic tweezers ,Optical Tweezers ,RecQ helicase ,Protein domain ,DNA, Single-Stranded ,Plasma protein binding ,Biology ,medicine.disease_cause ,DNA-binding protein ,Magnetics ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,stomatognathic system ,Escherichia coli ,Genetics ,medicine ,Molecular Biology ,chemistry.chemical_classification ,RecQ Helicases ,Escherichia coli Proteins ,nutritional and metabolic diseases ,Cell biology ,DNA-Binding Proteins ,stomatognathic diseases ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Enzyme ,chemistry ,Nucleic Acid Conformation ,DNA ,Protein Binding - Abstract
The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ–SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB–RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ–DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.
- Published
- 2017
34. Robust fluorescent labelling of micropipettes for use in fluorescence microscopy: application to the observation of a mosquito borne parasite infection
- Author
-
Photini Sinnis, Amanda E. Balaban, Robert S. Balaban, and Keir C. Neuman
- Subjects
0301 basic medicine ,Histology ,Enamel paint ,Chemistry ,Pipette ,Nanotechnology ,Intravital Imaging ,Fluorescence ,Pathology and Forensic Medicine ,03 medical and health sciences ,Fluorescent labelling ,030104 developmental biology ,visual_art ,Microscopy ,visual_art.visual_art_medium ,Fluorescence microscope ,Biophysics ,Parasite hosting - Abstract
Summary The ability to monitor micropipette injections with a high-resolution fluorescent microscope has utility for a variety of applications. Herein, different approaches were tested for creating broad-band fluorescently labelled glass micropipettes including: UV cured glass glues, baked glass enamel containing fluorescent dyes as well as nanodiamonds attached during pipette formation in the microforge. The most robust and simplest approach was to use labelled baked enamel on the exterior of the pipette. This approach was tested using pipettes designed to mimic a mosquito proboscis for the injection of the malaria parasite, Plasmodium spp., into the dermis of a living mouse ear. The pipette (∼30 micron diameter) was easily detected in the microscopy field of view and tolerated multiple insertions through the skin. This simple inexpensive approach to fluorescently labelling micropipettes will aid in the development of procedures under the fluorescent microscope.
- Published
- 2017
35. Activities of gyrase and topoisomerase IV on positively supercoiled DNA
- Author
-
Sylvia A. McPherson, Rachel E. Ashley, Charles L. Turnbough, Neil Osheroff, Keir C. Neuman, and Andrew Dittmore
- Subjects
DNA Topoisomerase IV ,0301 basic medicine ,Topoisomerase IV ,Cleavage (embryo) ,medicine.disease_cause ,DNA gyrase ,03 medical and health sciences ,chemistry.chemical_compound ,Genetics ,medicine ,Escherichia coli ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Nucleic Acid Enzymes ,DNA, Superhelical ,Escherichia coli Proteins ,030104 developmental biology ,Enzyme ,chemistry ,DNA Gyrase ,Bacillus anthracis ,biology.protein ,Biophysics ,DNA supercoil ,DNA ,Cytokinesis - Abstract
Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.
- Published
- 2017
36. Brownian ratchet mechanisms of ParA-mediated partitioning
- Author
-
Anthony G. Vecchiarelli, Keir C. Neuman, Kiyoshi Mizuuchi, Jian Liu, and Longhua Hu
- Subjects
Adenosine Triphosphatases ,0301 basic medicine ,Physics::Biological Physics ,Protein Conformation ,Brownian ratchet ,Biology ,Article ,Diffusion ,Quantitative Biology::Subcellular Processes ,Kinetics ,03 medical and health sciences ,030104 developmental biology ,Classical mechanics ,Models, Chemical ,Mathematics::Probability ,Physics::Atomic and Molecular Clusters ,Physics::Chemical Physics ,Molecular Biology ,Plasmids - Abstract
• Recent investigations have converged upon a new model in which ParA-type partitions as a Brownian ratchet.
- Published
- 2017
37. madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy
- Author
-
Jason Yi, Valarie A. Barr, Keir C. Neuman, Lawrence E. Samelson, Jennifer Hong, and Asit Kumar Manna
- Subjects
0301 basic medicine ,Receptor complex ,Statistics as Topic ,Receptors, Antigen, T-Cell ,Fluorescent Antibody Technique ,Image processing ,Articles ,Cell Biology ,Biology ,Fluorescence ,Multiplexing ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Microscopy, Fluorescence ,Microscopy ,Image Processing, Computer-Assisted ,Methods ,Molecule ,Fiducial marker ,Biological system ,Molecular Biology ,030217 neurology & neurosurgery ,Heterogeneous network ,Fluorescent Dyes - Abstract
A highly multiplexed superresolution imaging strategy with single-molecule accuracy enabled by fluorescent nanodiamonds called madSTORM affords the ability to define spatial relationships among constituent molecules within structures. It makes it possible to probe the molecular topology of complex signaling cascades and other heterogeneous networks., Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and
- Published
- 2016
38. Author response: Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase
- Author
-
Keir C. Neuman, Mihály Kovács, Gábor M. Harami, and Yeonee Seol
- Subjects
Physics ,Sequence dependent ,RecQ helicase ,Computational biology ,Homology (biology) - Published
- 2019
39. Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase
- Author
-
Keir C. Neuman, Yeonee Seol, Gábor M. Harami, and Mihály Kovács
- Subjects
0301 basic medicine ,Magnetic tweezers ,QH301-705.5 ,RecQ helicase ,Science ,Structural Biology and Molecular Biophysics ,homologous recombination ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Biochemistry and Chemical Biology ,Escherichia coli ,A-DNA ,Biology (General) ,unwinding mechanism ,Recombination, Genetic ,General Immunology and Microbiology ,biology ,RecQ Helicases ,General Neuroscience ,E. coli ,Helicase ,General Medicine ,DNA ,single molecule biophysics ,Kinetics ,helicase ,030104 developmental biology ,Structural biology ,chemistry ,biology.protein ,Biophysics ,Medicine ,Homologous recombination ,magnetic tweezers ,030217 neurology & neurosurgery ,genome stability ,Research Article - Abstract
RecQ helicases promote genomic stability through their unique ability to suppress illegitimate recombination and resolve recombination intermediates. These DNA structure-specific activities of RecQ helicases are mediated by the helicase-and-RNAseD like C-terminal (HRDC) domain, via unknown mechanisms. Here, employing single-molecule magnetic tweezers and rapid kinetic approaches we establish that the HRDC domain stabilizes intrinsic, sequence-dependent, pauses of the core helicase (lacking the HRDC) in a DNA geometry-dependent manner. We elucidate the core unwinding mechanism in which the unwinding rate depends on the stability of the duplex DNA leading to transient sequence-dependent pauses. We further demonstrate a non-linear amplification of these transient pauses by the controlled binding of the HRDC domain. The resulting DNA sequence- and geometry-dependent pausing may underlie a homology sensing mechanism that allows rapid disruption of unstable (illegitimate) and stabilization of stable (legitimate) DNA strand invasions, which suggests an intrinsic mechanism of recombination quality control by RecQ helicases., eLife digest Molecules of DNA carry instructions for all of the biological processes that happen in cells. Therefore, it is very important that cells maintain their DNA and quickly repair any damage. DNA molecules are made of two strands that twist together to form a double helix. The most reliable way to repair damage affecting both DNA strands involves a process known as homologous recombination. In this process, one of the strands of the broken DNA joins up with a strand of an identical or similar DNA molecule to make a triple-stranded structure known as a D-loop. This allows the cell to rebuild the damaged DNA using the intact DNA as a template. To ensure that the DNA is repaired correctly, enzymes known as RecQ helicases bind to and unwind D-loops if the strand pairs are poorly matched, whilst not disrupting pairs that are correctly matched. It remains unclear, however, how these enzymes are able to distinguish whether DNA strands in D-loops are a good or bad pair. To address this question, Seol, Harami et al. measured how individual RecQ helicases from a bacterium known as Escherichia coli unwind DNA. The experiments showed that the enzymes were better able to unwind sections of double-stranded DNA that were less stable than other sections of DNA (indicating the two strands may be a bad match). This causes the helicase to pause at stable sections of the DNA as it unwinds the double helix of the D-loop. Further experiments showed that a region of the helicase known as the HRDC domain increased the duration of these pauses, leading to a dramatic decrease in the unwinding speed. Seol, Harami et al. propose that this difference in unwinding speed prevents RecQ from unwinding legitimate matching D-loops while permitting rapid disruption of illegitimate D-loops that could lead to damaged DNA being repaired incorrectly. Mutations in the human versions of RecQ helicases lead to Bloom’s syndrome and Werner’s syndrome in which individuals are predisposed to developing cancer. Understanding how cells repair DNA may ultimately help to treat individuals with these and other similar conditions.
- Published
- 2019
40. Polydopamine encapsulation of fluorescent nanodiamonds for biomedical applications
- Author
-
Paul A. Roche, Yasuharu Takagi, Hak-Sung Jung, Kyung-Jin Cho, Yeonee Seol, Keir C. Neuman, and Andrew Dittmore
- Subjects
Streptavidin ,Materials science ,Bioconjugation ,Biocompatibility ,Nanoparticle ,Nanotechnology ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,Article ,0104 chemical sciences ,Electronic, Optical and Magnetic Materials ,Nanoclusters ,Biomaterials ,chemistry.chemical_compound ,chemistry ,Biotinylation ,Drug delivery ,Electrochemistry ,Surface modification ,0210 nano-technology - Abstract
Fluorescent nanodiamonds (FNDs) are promising bio-imaging probes compared with other fluorescent nanomaterials such as quantum dots, dye-doped nanoparticles, and metallic nanoclusters, due to their remarkable optical properties and excellent biocompatibility. Nevertheless, they are prone to aggregation in physiological salt solutions, and modifying their surface to conjugate biologically active agents remains challenging. Here, inspired by the adhesive protein of marine mussels, we demonstrate encapsulation of FNDs within a polydopamine (PDA) shell. These PDA surfaces are readily modified via Michael addition or Schiff base reactions with molecules presenting thiol or nitrogen derivatives. We describe modification of PDA shells by thiol terminated poly(ethylene glycol) (PEG-SH) molecules to enhance colloidal stability and biocompatibility of FNDs. We demonstrate their use as fluorescent probes for cell imaging; we find that PEGylated FNDs are taken up by HeLa cells and mouse bone marrow-derived dendritic cells and exhibit reduced nonspecific membrane adhesion. Furthermore, we demonstrate functionalization with biotin-PEG-SH and perform long-term high-resolution single-molecule fluorescence based tracking measurements of FNDs tethered via streptavidin to individual biotinylated DNA molecules. Our robust polydopamine encapsulation and functionalization strategy presents a facile route to develop FNDs as multifunctional labels, drug delivery vehicles, and targeting agents for biomedical applications.
- Published
- 2019
41. Kinetic Pathway of Torsional DNA Buckling
- Author
-
Andrew Dittmore, Jonathan Silver, and Keir C. Neuman
- Subjects
0301 basic medicine ,Magnetic tweezers ,Optical Tweezers ,Kinetic energy ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Metastability ,0103 physical sciences ,Materials Chemistry ,Torque ,Physical and Theoretical Chemistry ,010306 general physics ,Buckle ,Physics ,Condensed matter physics ,Torsional buckling ,DNA ,Surfaces, Coatings and Films ,Kinetics ,030104 developmental biology ,Buckling ,chemistry ,Thermodynamics - Abstract
In magnetic tweezers experiments, we observe that torsional DNA buckling rates and transition state distances are insensitive to base-pairing defects. This is surprising because defects are expected to kink DNA and lower the energy of a localized loop. Nonetheless, base-pairing defects lead to pinning of buckled structures at the defects, which may be important for DNA repair in vivo. We find that the decrease in entropy from pinning roughly balances the decrease in bending energy, explaining why defects have little effect on buckling rates. Our data are generally consistent with elastic rod theory, which predicts that the transition-state structure for torsional buckling is a localized wave with a specific shape (“soliton”). The transition-state soliton decays to a metastable looped intermediate (“curl”) that is separated from the final, fully-buckled state by a second, low energy, barrier. DNAs with base mismatch defects buckle at lower torque, where elastic rod theory predicts the loop structure is more stable, and manifest an intermediate buckling structure consistent with such a loop. We estimate that, under our high force, high salt experimental conditions, the soliton barrier is approximately 10 k(B)T and, to reach this transition state from the unbuckled state, the system torque instantaneously decreases by approximately 1 pN·nm for DNA with or without a small defect.
- Published
- 2018
42. Cholesterol in Cargo Membrane Amplifies inhibitory Effect of Map Tau On Kinesin-1
- Author
-
Jing Xu, Michael Vershinin, James Ferrare, Keir C. Neuman, Qiaochu Li, Jonathan Silver, Weihong Qiu, John O. Wilson, and Stephen J. King
- Subjects
chemistry.chemical_compound ,Membrane ,chemistry ,Cholesterol ,Biophysics ,Kinesin ,Inhibitory effect - Published
- 2021
43. Mapping the Interplay between DNA Topology and Sequence on Type II Topoisomerase Binding and Cleavage Activity
- Author
-
Keir C. Neuman, Rachel Kim, and Shannon J. Mckie
- Subjects
chemistry.chemical_compound ,Chemistry ,Stereochemistry ,Biophysics ,Cleavage (embryo) ,Type II topoisomerase ,Topology (chemistry) ,DNA ,Sequence (medicine) - Published
- 2021
44. Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling
- Author
-
Keir C. Neuman, Jonathan Silver, Barry L. Marmer, Andrew Dittmore, Susanta K. Sarkar, and Gregory I. Goldberg
- Subjects
Tail ,0301 basic medicine ,Materials science ,Fibrillar Collagens ,Proteolysis ,Context (language use) ,macromolecular substances ,Plasma protein binding ,Matrix metalloproteinase ,010402 general chemistry ,Fibril ,Cleavage (embryo) ,01 natural sciences ,Tendons ,Extracellular matrix ,03 medical and health sciences ,medicine ,Animals ,Mechanical Phenomena ,Multidisciplinary ,medicine.diagnostic_test ,Biological Sciences ,Matrix Metalloproteinases ,Single Molecule Imaging ,Extracellular Matrix ,Rats ,0104 chemical sciences ,Crystallography ,030104 developmental biology ,Biophysics ,Collagenase ,Protein Binding ,medicine.drug - Abstract
Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal-strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.
- Published
- 2016
45. Domain Rigidity Modulates the Catalytic Activity of E. coli Type IA DNA Topoisomerases
- Author
-
Yuk-Ching Tse Dinh, Yeonee Seol, and Keir C. Neuman
- Subjects
chemistry.chemical_compound ,Rigidity (electromagnetism) ,biology ,chemistry ,Topoisomerase ,Domain (ring theory) ,Biophysics ,biology.protein ,DNA ,Catalysis - Published
- 2020
46. Coarse-Grained Modeling of DNA Plectoneme Formation in the Presence of Base-Pair Mismatches
- Author
-
Keir C. Neuman, Parth Rakesh Desai, and Siddhartha Das
- Subjects
Molecular dynamics ,chemistry.chemical_compound ,Materials science ,chemistry ,Base pair ,Chemical physics ,Dynamics (mechanics) ,Biophysics ,DNA supercoil ,Bending ,Coarse-grained modeling ,DNA ,Nucleobase - Abstract
Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting mean-field theory to study the effect of mismatched base pairs on DNA supercoiling. The coarse-grained approach reproduces experimentally observed deterministic plectoneme pinning at the mismatch under conditions of relatively high force (> 2 pN) and high salt concentration (> 0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. The simulation results broadly agree with existing statistical mechanical mean-field theoretical predictions for the high force-high salt regime. The coarse-grained simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.
- Published
- 2020
47. Probing and Visualization of the RecQ Helicase-Induced DNA Binding Mode Change of the Bacterial Single-Stranded DNA Binding (SSB) Protein
- Author
-
Keir C. Neuman, Veronika Baráth, Lili Farkas, Mihály Kovács, Yeonee Seol, Gábor M. Harami, Ágnes Hubert, and Zoltán Kovács
- Subjects
chemistry.chemical_compound ,biology ,Chemistry ,RecQ helicase ,Single-stranded DNA binding ,Biophysics ,biology.protein ,Mode change ,DNA ,Single-stranded binding protein - Published
- 2020
48. Defect-Facilitated Buckling in Supercoiled Double-Helix DNA
- Author
-
Sumitabha Brahmachari, Andrew Dittmore, John F. Marko, Keir C. Neuman, and Yasuharu Takagi
- Subjects
0301 basic medicine ,Materials science ,Nucleation ,Ionic bonding ,Thermal fluctuations ,01 natural sciences ,Molecular physics ,Article ,03 medical and health sciences ,symbols.namesake ,0103 physical sciences ,Molecule ,010306 general physics ,Scaling ,030304 developmental biology ,Physics ,Quantitative Biology::Biomolecules ,0303 health sciences ,DNA, Superhelical ,030104 developmental biology ,Torque ,Buckling ,symbols ,DNA supercoil ,Salts ,Hamiltonian (quantum mechanics) - Abstract
We present a statistical-mechanical model for stretched twisted double-helix DNA, where thermal fluctuations are treated explicitly from a Hamiltonian without using any scaling hypotheses. Our model applied to defect-free supercoiled DNA describes the coexistence of multiple plectoneme domains in long DNA molecules at physiological salt concentrations ($\ensuremath{\approx}0.1\phantom{\rule{0.16em}{0ex}}\mathrm{M} {\mathrm{Na}}^{+}$) and stretching forces ($\ensuremath{\approx}1\phantom{\rule{0.16em}{0ex}}\mathrm{pN})$. We find a higher (lower) number of domains at lower (higher) ionic strengths and stretching forces, in accord with experimental observations. We use our model to study the effect of an immobile point defect on the DNA contour that allows a localized kink. The degree of the kink is controlled by the defect size, such that a larger defect further reduces the bending energy of the defect-facilitated kinked end loop. We find that a defect can spatially pin a plectoneme domain via nucleation of a kinked end loop, in accord with experiments and simulations. Our model explains previously reported magnetic tweezer experiments [A. Dittmore et al., Phys. Rev. Lett. 119, 147801 (2017)] showing two buckling signatures: buckling and ``rebuckling'' in supercoiled DNA with a base-unpaired region. Comparing with experiments, we find that under 1 pN force, a kinked end loop nucleated at a base-mismatched site reduces the bending energy by $\ensuremath{\approx}0.7 {k}_{B}T$ per unpaired base. Our model predicts the coexistence of three states at the buckling and rebuckling transitions, which warrants new experiments.
- Published
- 2018
49. Evidence for a Solenoid Phase of Supercoiled DNA
- Author
-
Andrew Dittmore and Keir C. Neuman
- Subjects
Physics ,Physics::Biological Physics ,Quantitative Biology::Biomolecules ,FOS: Physical sciences ,Biomolecules (q-bio.BM) ,Condensed Matter - Soft Condensed Matter ,Quantitative Biology::Genomics ,chemistry.chemical_compound ,Classical mechanics ,Quantitative Biology - Biomolecules ,chemistry ,Cascade ,Biological Physics (physics.bio-ph) ,FOS: Biological sciences ,DNA supercoil ,Soft Condensed Matter (cond-mat.soft) ,Physics - Biological Physics ,Constant force ,DNA ,Writhe - Abstract
In mechanical manipulation experiments, a single DNA molecule overwound at constant force undergoes a discontinuous drop in extension as it buckles and forms a superhelical loop (a plectoneme). Further overwinding the DNA, we observe an unanticipated cascade of highly regular discontinuous extension changes associated with stepwise plectoneme lengthening. This phenomenon is consistent with a model in which the force-extended DNA forms barriers to plectoneme lengthening caused by topological writhe. Furthermore, accounting for writhe in a fluctuating solenoid gives an improved description of the measured force-dependent effective torsional modulus of DNA, providing a reliable formula to estimate DNA torque. Our data and model thus provide context for further measurements and theories that capture the structures and mechanics of supercoiled biopolymers., Comment: See https://www.biorxiv.org/content/biorxiv/suppl/2018/04/16/302661.DC1/302661-1.pdf for supporting information and https://drive.google.com/open?id=1sd6j4P-lYrZpgPbPAA1N8z2yWG2kLeQT for code
- Published
- 2018
- Full Text
- View/download PDF
50. A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force
- Author
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Anthony G. Vecchiarelli, Yeonee Seol, Keir C. Neuman, and Kiyoshi Mizuuchi
- Subjects
intracellular transport ,Short Communications ,plasmid partition ,Biology ,subcellular organization ,Bacterial cell structure ,Protein filament ,Motor protein ,chemistry.chemical_compound ,Structural Biology ,Organelle ,Nucleoid ,Cytoskeleton ,Mitosis ,Mechanical Phenomena ,Adenosine Triphosphatases ,Organelles ,Biological Transport ,Cell Biology ,General Medicine ,Cell biology ,ParA ATPase ,chemistry ,bacterial chromosome segregation ,DNA ,Plasmids - Abstract
DNA segregation is a critical process for all life, and although there is a relatively good understanding of eukaryotic mitosis, the mechanism in bacteria remains unclear. The small size of a bacterial cell and the number of factors involved in its subcellular organization make it difficult to study individual systems under controlled conditions in vivo. We developed a cell-free technique to reconstitute and visualize bacterial ParA-mediated segregation systems. Our studies provide direct evidence for a mode of transport that does not use a classical cytoskeletal filament or motor protein. Instead, we demonstrate that ParA-type DNA segregation systems can establish a propagating ParA ATPase gradient on the nucleoid surface, which generates the force required for the directed movement of spatially confined cargoes, such as plasmids or large organelles, and distributes multiple cargos equidistant to each other inside cells. Here we present the critical principles of our diffusion-ratchet model of ParA-mediated transport and expand on the mathematically derived chemophoresis force using experimentally-determined biochemical and cellular parameters.
- Published
- 2015
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