Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.
In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRI(E111Q), lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Ilya J
IJ
Finkelstein
Mari-Liis
ML
Visnapuu
Eric C
EC
Greene
D010582:Bacteriophage lambda / Q000235:genetics; D055162:Biocatalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009068:Movement; D009707:Nucleosomes / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D045663:Quantum Dots; D013997:Time Factors
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D056968:Lac Repressors; D009707:Nucleosomes; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D043211:Exodeoxyribonuclease V; D015246:Deoxyribonuclease EcoRI