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Updated: Mar 28, 2026

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation
Takuma Iwasa1,2, Yong-Woon Han1,3, Ryo Hiramatsu4
1Institute for Integrated Cell-Materials Science (WPI-iCeMS), Kyoto University, Sakyo, Kyoto 606-8501, Japan.
Abstract:
The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.
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