Loading dynamics of a sliding DNA clamp.
Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.
Department of Physics, School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang (Korea).
Won-Ki
WK
Cho
Slobodan
S
Jergic
Daehyung
D
Kim
Nicholas E
NE
Dixon
Jong-Bong
JB
Lee
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis
D002232:Carbocyanines; D029968:Escherichia coli Proteins; C085321:cyanine dye 5; D000255:Adenosine Triphosphate; D004247:DNA
DNA clamps; DNA replication; single-molecule FRET; single-molecule polarization; ternary complexes