Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules
| Autor: | Brendon McGuinness, David Levens, Kimberly Metera, Craig J. Benham, Shane Scott, Sabrina Leslie, Cynthia Shaheen, Fedor Kouzine |
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| Rok vydání: | 2019 |
| Předmět: |
genetic structures
02 engineering and technology Biology 010402 general chemistry 01 natural sciences Polyethylene Glycols 03 medical and health sciences chemistry.chemical_compound Plasmid Transcription (biology) Genetics Molecular Biology 030304 developmental biology 0303 health sciences DNA Superhelical Chemistry Oligonucleotide Osmolar Concentration DNA replication 021001 nanoscience & nanotechnology Crowding Single Molecule Imaging 0104 chemical sciences Ionic strength Biophysics DNA supercoil 0210 nano-technology Oligonucleotide Probes DNA Plasmids |
| Zdroj: | Nucleic Acids Research |
| DOI: | 10.1101/591008 |
| Popis: | DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe–plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe–plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNA’s proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe–plasmid interactions once the site is unwound. |
| Databáze: | OpenAIRE |
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