Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.
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http://dx.doi.org/10.3791/61320 | DOI Listing |
Methods Mol Biol
December 2024
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA.
Homologous recombination (HR) is the principal pathway undertaken by a cell for the error-free repair of DNA double-strand breaks that are frequently encountered by the cell. HR can be initiated at the sites of DNA double-strand breaks by generating long stretches of single-stranded 3' DNA overhang through a process called DNA end resection. In one DNA end resection pathway, this is achieved via the concerted effort of specialized machinery involving the RecQ family helicase BLM, the helicase/endonuclease DNA2, and a single-strand DNA binding protein complex RPA.
View Article and Find Full Text PDFBMB Rep
December 2024
Department of Physics and Astronomy, University of Texas Rio Grande Valley, Edinburg, Texas 78539, USA.
Single-molecule techniques allow researchers to investigate individual molecules and obtain unprecedented details of the heterogeneous nature of biological entities. They play instrumental roles in studying DNA-protein interactions due to the ability to visualize DNA or proteins and to manipulate individual DNA molecules by applying force or torque. Here, we describe single-molecule DNA-flow stretching assays as hybrid tools that combine forces with fluorescence.
View Article and Find Full Text PDFSci Total Environ
October 2024
School of Biological & Environmental Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK. Electronic address:
Environmental DNA (eDNA) analysis has now become a core approach in marine biodiversity research, which typically involves the collection of water or sediment samples. Yet, recently, filter-feeding organisms have received much attention for their potential role as natural eDNA samplers. While the indiscriminate use of living organisms as 'sampling tools' might in some cases raise conservation concerns, there are instances in which highly abundant sessile organisms may become a nuisance as biofouling on artificial marine structures.
View Article and Find Full Text PDFBiochemistry (Mosc)
April 2024
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Chromatin is an epigenetic platform for implementation of DNA-dependent processes. Nucleosome, as a basic level of chromatin compaction, largely determines its properties and structure. In the study of nucleosomes structure and functions physicochemical tools are actively used, such as magnetic and optical "tweezers", "DNA curtains", nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy, as well as optical methods based on Förster resonance energy transfer.
View Article and Find Full Text PDFMethods Cell Biol
February 2024
Gene Center Munich, Ludwig Maximilian University, Munich, Germany. Electronic address:
Phase separation is one key mechanism to organize chromatin into compartments and to regulate the activity of the genome. The formation of liquid-like droplets within the nucleus is driven by protein association to the DNA via multivalent binding and the recruitment of other proteins building a concentrated reaction environment. Common methods to study phase separation and its liquid-like nature are based on microscopy of the formed droplets but lack the resolution to obtain information on the molecular level.
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