Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers.
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.
Mameren
Joost van
Jv
Modesti
Mauro
M
Kanaar
Roland
R
Wyman
Claire
C
Wuite
Gijs J L
GJ
Peterman
Erwin J G
EJ
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*
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase