Difference: ProjectDescription (2 vs. 3)

We are interested in elucidating the mechanisms underlying how Rad51-DNA filaments find and align homologous DNA, and also catalyze the repair process. We hypothesize that the nucleoprotein filament searches dsDNA using a translocation mechanism driven by the Snf2 motor proteins Rad54 or Rdh54, and that a second function of these motors is to clear the dsDNA of bound proteins such as nucleosomes.

To test our hypotheses, we use a unique microscopy system able to study recombination at the level of single molecules. Total internal reflection fluorescence microscopy (TIRFM) can directly image DNA repair complexes interacting with dsDNA in real time. To perform TIRFM, a laser is focused through a microscope slide and reflects off the slide-buffer interface. While most of the beam is reflected away from the interface, some of the beam penetrates the interface and illuminates a very small volume on the sample side of the slide, typically penetrating 100-300 nm into the aqueous medium. Single fluorescent molecules very close to the slide surface can be observed using this technique.

Flow cells used in this project are covered with a lipid bilayer, preventing nonspecific interactions with the surface and also able to support ordered arrays of tethered DNA molecules. DNA molecules are biotinylated at one end, and the flow cell surface is sparsely coated with neutravidin. The biotinylated DNA is then assembled onto the surface with the neutravidin molecules providing anchors to the flow cell surface. Tethered DNA molecules are then subjected to a buffer flow, which extends the DNA along the surface, making their entire length within the sample volume illuminated by TIRFM.

In our experiments, DNA is made fluorescent in 2 ways. First, the intercalating dye YOYO1 can be introduced at low concentrations into the duplex. Alternatively, the end of the DNA that is not biotinylated can be modified with a digoxigenin, which leaves the duplex in a more native state. Individual quantum dots attached to anti-digoxigenin antibodies are then used to identify the ends of DNA molecules.

In this project, we propose to form stable Rad51 filaments on ssDNA and inject these into dsDNA-containing flow cells. If both the dsDNA and the ssDNA of the nucleoprotein filaments are fluorescently labeled, we will be able to follow the recombination reaction in real time. We wish to use NYSBC’s EM facilities to visualize quantum dots as well as DNA molecules bound to quantum dots to demonstrate that we are able to make these reagents and to determine the stoichiometry of DNA molecules bound to individual quantum dots.