Watching individual proteins acting on single molecules of DNA.

Ichiro Amitani, Bian Liu, Christopher C. Dombrowski, Ronald J. Baskin, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

Abstract

In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.

Original languageEnglish (US)
Pages (from-to)261-291
Number of pages31
JournalMethods in Enzymology
Volume472
StatePublished - Jan 1 2010
Externally publishedYes

Fingerprint

Molecules
DNA
Proteins
Optical Tweezers
Microfluidics
Fluorescence microscopy
Polystyrenes
Microspheres
Fluorescence Microscopy
DNA Repair
Design of experiments
Labeling
Genetic Recombination
Microscopy
Microscopic examination
Lasers
Repair
Research Design
Visualization
Maintenance

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Amitani, I., Liu, B., Dombrowski, C. C., Baskin, R. J., & Kowalczykowski, S. C. (2010). Watching individual proteins acting on single molecules of DNA. Methods in Enzymology, 472, 261-291.

Watching individual proteins acting on single molecules of DNA. / Amitani, Ichiro; Liu, Bian; Dombrowski, Christopher C.; Baskin, Ronald J.; Kowalczykowski, Stephen C.

In: Methods in Enzymology, Vol. 472, 01.01.2010, p. 261-291.

Research output: Contribution to journalArticle

Amitani, I, Liu, B, Dombrowski, CC, Baskin, RJ & Kowalczykowski, SC 2010, 'Watching individual proteins acting on single molecules of DNA.', Methods in Enzymology, vol. 472, pp. 261-291.
Amitani I, Liu B, Dombrowski CC, Baskin RJ, Kowalczykowski SC. Watching individual proteins acting on single molecules of DNA. Methods in Enzymology. 2010 Jan 1;472:261-291.
Amitani, Ichiro ; Liu, Bian ; Dombrowski, Christopher C. ; Baskin, Ronald J. ; Kowalczykowski, Stephen C. / Watching individual proteins acting on single molecules of DNA. In: Methods in Enzymology. 2010 ; Vol. 472. pp. 261-291.
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