Watching individual proteins acting on single molecules of DNA.
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.
Amitani
Ichiro
I
Department of Microbiology, University of California, Davis, California, USA.
Liu
Bian
B
Dombrowski
Christopher C
CC
Baskin
Ronald J
RJ
Kowalczykowski
Stephen C
SC
D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry; D052898:Optical Tweezers; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry / Q000378:metabolism; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; C089187:cyanine dye 3; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D043211:Exodeoxyribonuclease V