NSF PRFB FY 2023: Deciphering the Structural Evolution of Alternative-End Joining

This action funds an NSF Postdoctoral Research Fellowship in Biology for FY 2023, Integrative Research Investigating the Rules of Life Governing Interactions Between Genomes, Environment, and Phenotypes. The fellowship supports research and training of the fellow that will contribute to the area of Rules of Life in innovative ways. DNA is the fundamental genetic storage molecule for all cellular life on Earth, yet it is susceptiple to damage by various cellular and environmental processes, which can impair cell survival. Consequently, cells have evolved dedicated DNA repair pathways, collectively known as the DNA damage response (DDR) to recognize and correct mistakes in the genome. When both strands of the DNA double helix are broken, this creates a double-strand break (DSB), and is repaired by a mechanisms called alternative-end joining (alt-EJ), a pathway that stitches two DNA ends back together. The structural basis for how this process works is unknown, but it is important for the evolution of all complex life on Earth. This research will explore the 3-dimensional architecture of proteins and DNAs within the alt-EJ pathway, and will provide a deeper understanding for how organisms protect and repair their genome from damage. This research will also support teaching, mentoring, and outreach by the fellow to improve scientific communication and participation of underrepresented groups. The alternative-end joining (alt-EJ) pathway is a multi-step pathway that is conducted by the DNA helicase/polymerase, Pol theta (Polθ). Alt-EJ is an intermediate DSB repair pathway between non-homologous end-joining (NHEJ) and homologous recombination (HR), and this research will contribute to the understanding of pathway choice. Structurally, full-length Polθ has not been observed in its native context, and the recruitment of Polθ to DNA and coordination with other repair pathways has not been elucidated. The research goals of this proposal involve coupling high-resolution cryogenic-electron microscopy (cryo-EM) and biochemical/phylogenetic techniques to uncover two objectives: (i) the structural basis for human alternative-end joining by Polθ and (ii) the mechanistic evolution of Polθ and alt-EJ using biochemical assays and molecular phylogeny. Alt-EJ complexes will be assembled on magnetic DNA-beads to construct specific macromolecular assemblies for visualization at atomic resolution. The proposed research will provide the fellow with training and development opportunities in structural biology, biochemistry/biophysics, and scientific communication (publications, conferences, seminars). The fellow will also continue to gain experience in teaching and mentoring undergraduate and graduate students, and work to improve diversity and inclusivity in the STEM fields by participation in DEI and implicit bias training. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.