Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers

  • Van Mameren J
  • Modesti M
  • Kanaar R
 et al. 
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Abstract

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. © 2006 by the Biophysical Society.

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Authors

  • Mauro ModestiCentre de Recherche en Cancerologie de Marseille

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  • Joost Van Mameren

  • Roland Kanaar

  • Claire Wyman

  • Gijs J L Wuite

  • Erwin J G Peterman

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