title: Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers.
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 approximately 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.
authors.forename: Joost van
authors.initials: Jv
authors.lastname: Mameren
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.forename: Claire
authors.initials: C
authors.lastname: Wyman
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
authors.forename: Erwin J G
authors.initials: EJ
authors.lastname: Peterman
mesh_terms: D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004548:Elasticity; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D052898:Optical Tweezers; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase