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Dissecting Elastic Heterogeneity along DNA Molecules Coated Partly with Rad51 Using Concurrent Fluorescence Microscopy and Optical Tweezers

Joost van Mameren ^*, Mauro Modesti , Roland Kanaar  , Claire Wyman  , Gijs J. L. Wuite ^* and Erwin J. G. Peterman ^*

^* Laser Centre and Department of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands; Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; and Department of Radiation Oncology, Erasmus Medical Center Daniël den Hoed Cancer Center, Rotterdam, The Netherlands

Correspondence: Address reprint requests and inquiries to Gijs Wuite, Tel.: 31-20-5987987; E-mail: gwuite{at}nat.vu.nl.

Nucleoprotein filament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51 filament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein filaments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well-known (over)stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.

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