Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

19-POS Board 19 DYNAMICS OF THE INTERACTION BETWEEN THE BACTERIAL NHEJ REPAIR PROTEINS KU AND LIGD AND DNA STUDIED USING SINGLE MOLECULE NANOFLUIDICS Evgeniya Pavlova 1 ; Anusha Budida 1 ; Robin Öz 1 ; Fredrik Westerlund 1 ; 1 Chalmers University of Technology, Department of Biology and Biological Engineering, Göteborg, Sweden The non-homologous end-joining (NHEJ) pathway for repair of DNA double-stranded breaks was only recently shown to exist in some prokaryotes, where its machinery is minimal. For Bacillus Subtilis the homodimer Ku and the Ligase D (LigD) have been shown both in vitro and in vivo to be the only two essential factors. The mechanism of the LigD/Ku/DNA complex formation has not been studied in detail, in particular on the single DNA molecule level. Uncovering the molecular details of the process would open possibilities for future development of antibiotics that target bacterial NHEJ. In this work, we visualize and characterize interactions between the bacterial NHEJ proteins and DNA on the single DNA molecule level.Since NHEJ occurs on DNA ends, traditional single molecule techniques, such as magnetic and optical tweezers that require the DNA molecule to be attached to a surface of a bead, are challenging to use. We instead use a nanofluidic setup, in which the DNA is stretched only due to confinement in nanochannels. By mixing Ku with λ -phage DNA and confining the samples in nanochannels we show that Ku alone brings DNA ends together to form DNA circles and concatemers in a concentration and DNA/protein ratio dependent manner. By performing EMSA we demonstrate that DNA-binding of Ku is cooperative and that LigD stabilizes DNA end-joining by Ku. Moreover, by performing a ligation reaction and imaging the sample 4 hours post reaction we show that Ku remains bound to DNA after ligation has occurred, suggesting a system is available in vivo for removing Ku when ligation is completed. Together these results open opportunities for antibiotic research, e.g. novel drugs that target Ku-DNA or LigD-Ku interactions.

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