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

该行动资助了美国国家科学基金会2023财年生物学博士后研究奖学金，研究基因组，环境和表型之间相互作用的生命规则的综合研究。该奖学金支持将以创新方式对生活规则领域作出贡献的研究员的研究和培训。DNA是地球上所有细胞生命的基本遗传储存分子，但它很容易受到各种细胞和环境过程的破坏，从而影响细胞的生存。因此，细胞进化出了专门的DNA修复途径，统称为DNA损伤反应（DDR），以识别和纠正基因组中的错误。当DNA双螺旋的两条链都断裂时，就会产生双链断裂（DSB），并通过一种称为替代末端连接（alt-EJ）的机制进行修复，这是一种将两个DNA末端重新缝合在一起的途径。这个过程的结构基础尚不清楚，但它对地球上所有复杂生命的进化都很重要。本研究将探索alt-EJ通路中蛋白质和dna的三维结构，并将为生物体如何保护和修复其基因组免受损伤提供更深入的理解。这项研究还将支持该研究员的教学、指导和推广工作，以改善代表性不足群体的科学交流和参与。替代端连接（alt-EJ）途径是由DNA解旋酶/聚合酶Polθ (Polθ)进行的多步骤途径。Alt-EJ是介于非同源末端连接（non-homologous end-joining， NHEJ）和同源重组（homologous recombination， HR）之间的DSB修复中间通路，本研究将有助于对通路选择的理解。在结构上，全长Polθ在其天然环境中未被观察到，Polθ在DNA上的招募以及与其他修复途径的协调也未被阐明。本提案的研究目标涉及高分辨率低温电子显微镜（cryo-EM）和生物化学/系统发育技术的结合，以揭示两个目标：(i)人类选择性末端连接Polθ的结构基础；（ii）利用生物化学分析和分子系统发育研究Polθ和alt-EJ的机制进化。Alt-EJ配合物将组装在磁性dna珠上，以构建特定的大分子组装，以便在原子分辨率下可视化。该研究将为研究员提供结构生物学、生物化学/生物物理学和科学传播（出版物、会议、研讨会）方面的培训和发展机会。该研究员还将继续获得教学和指导本科生和研究生的经验，并通过参与DEI和隐性偏见培训，努力提高STEM领域的多样性和包容性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.