Abstract

RecA plays a central role in homologous recombination of DNA. When RecA combines with dsDNA to form RecA-dsDNA nucleofilament, it unwinds dsDNA and changes its structure. The unwinding length extension of a DNA segment interacting with RecA has been studied by various techniques, but the dynamic differential stiffness of dsDNA conjugating with RecA has not been well characterized. We applied oscillatory optical tweezers to measure the differential stiffness of dsDNA molecules, interacting with RecA, as a function of time at a constant stretching force of 33.6pN. The values of the differential stiffness of DNA (for stretching force in the range of 20.0pN to 33.6pN) measured by oscillatory optical tweezers, both before and after its interaction with RecA, are consistent with those measured by stationary optical tweezers. In the dynamic measurement, we have shown that the association (or binding) rate increases with higher concentration of RecA; besides, we have also monitored in real-time the dissociation of RecA from the stretched RecA-dsDNA filament as ATPγS was washed off from the sample chamber. Finally, we verified that RecA (I26C), a form of RecA mutant, does not affect the differential stiffness of the stretched DNA sample. It implies that mutant RecA (I26C) does not bind to the DNA, which is consistent with the result obtained by conventional biochemical approach.

© 2009 OSA

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History
Original Manuscript: August 11, 2009
Manuscript Accepted: October 15, 2009
Revised Manuscript: October 14, 2009
Published: October 23, 2009

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Author Affiliations

Ming-Tzo Wei

Biophotonics Interdisciplinary Research Center, National Yang-Ming UniversityDepartment of Physics, Lehigh University, Bethlehem, Pennsylvania, 18015, USA

H. Daniel Ou-Yang

Department of Physics, Lehigh University, Bethlehem, Pennsylvania, 18015, USA

Chia-Hui Lien, Te- Yu Tseng

Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan

Arthur Chiou

Institute of Biophotonics, National Yang-Ming University, Taipei, TaiwanBiophotonics Interdisciplinary Research Center, National Yang-Ming University

Chien-Der Lee, Chung Wang, Ting-Fang Wang

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

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