Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g.

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.

Highly sensitive mapping of type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g.

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.

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.

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.

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 topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries.

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

ParABS partition systems, comprising the centromere-like DNA sequence , the -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 ParB and move by generating and following a local concentration gradient of nucleoid-bound ParA. However, the process through which ParB activates ParA-ATPase has not been defined.

Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase.

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.

Polydopamine encapsulation of fluorescent nanodiamonds for biomedical applications.

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.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

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.

RecQ helicase triggers a binding mode change in the SSB-DNA complex to efficiently initiate DNA unwinding.

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.

Combined Magnetic Tweezers and Micro-mirror Total Internal Reflection Fluorescence Microscope for Single-Molecule Manipulation and Visualization.

Magnetic tweezers is a versatile yet simple single-molecule manipulation technique that has been used to study a broad range of nucleic acids and nucleic acid-based molecular motors. In this chapter, we combine micro-mirror-based total internal reflection microscopy with a magnetic tweezers instrument, permitting simultaneous single-molecule visualization and mechanical manipulation. We provide a simple method to calibrate the evanescent wave penetration depth via supercoiling of DNA with a fluorescent nanodiamond-labeled magnetic bead and a complementary method employing a surface-immobilized fluorescent nanodiamond.

Distribution bias and biochemical characterization of TOP1MT single nucleotide variants.

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.

The dynamic interplay between DNA topoisomerases and DNA topology.

Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation.

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination.

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure.

The Dynamic Interplay Between DNA Topoisomerases and DNA Topology.

Topological properties of DNA influence its structure and biochemical interactions. Within the cell DNA topology is constantly in flux. Transcription and other essential processes including DNA replication and repair, alter the topology of the genome, while introducing additional complications associated with DNA knotting and catenation.

Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis.

Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student's t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo.

Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors.

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.

A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force.

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.

Poisoning of mitochondrial topoisomerase I by lamellarin D.

Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D.

Chiral discrimination and writhe-dependent relaxation mechanism of human topoisomerase IIα.

Background: Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils.

Results: Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity.

Conclusion: Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains.

Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification.

Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed.