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Visualizing Replication and Recombination on Single Molecules of DNA
Author(s) -
Kowalczykowski Steven
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.371.1
Subject(s) - dna , total internal reflection fluorescence microscope , biophysics , dna replication , chemistry , fluorescence microscope , microbiology and biotechnology , biology , nanotechnology , fluorescence , biochemistry , materials science , physics , quantum mechanics , membrane
It is now possible to image individual proteins acting on single molecules of DNA. Such imaging affords unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead, which is captured in an optical trap. The DNA is extended either by the force of solution flow in a micro‐flowcell, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases, translocation along DNA by motor proteins, self‐assembly of protein filaments on DNA as well as regulation of nucleation and growth, the search for DNA sequence homology protein‐DNA filaments, real‐time replication of DNA, and nucleosome structure and its remodeling. I will summarize how these experiments were done, and what we've learned from a selection of these experiments.