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Item Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes(Springer Science and Business Media LLC, 2023-10) Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Karunakaran, Simrithaa; Rau, Michael J.; Basore, Katherine; Pokhrel, Nilisha; Englander, Noah; Fitzpatrick, James A. J.; Bothner, Brian; Antony, EdwinHomologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.Item Highly-automated, high-throughput replication of yeast-based logic circuit design assessments(Oxford University Press, 2022-02) Goldman, Robert P; Moseley, Robert; Roehner, Nicholas; Cummins, Breschine; Vrana, Justin D; Clowers, Katie J; Bryce, Daniel; Beal, Jacob; DeHaven, Matthew; Nowak, Joshua; Higa, Trissha; Biggers, Vanessa; Lee, Peter; Hunt, Jeremy P.; Mosqueda, Lorraine; Haase, Steven B.; Weston, Mark; Zheng, George; Deckard, Anastasia; Gopaulakrishnan, Shweta; Stubbs, Joseph F.; Gaffney, Niall I.; Vaughn, Matthew W.; Maheshri, Narendra; Mikhalev, Ekaterina; Bartley, Bryan; Markeloff, Richard; Mitchell, Tom; Nguyen, Tramy; Sumorok, Daniel; Walczak, Nicholas; Myers, Chris; Zundel, Zach; Hatch, Benjamin; Scholz, James; Colonna-Romano, JohnWe describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency’s Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned.