NSF Postdoctoral Fellowship in Biology FY 2013

了解基因组的馆员：在过去的半个世纪里，人们对DNA加工酶和DNA聚合酶的构象变化进行了广泛的研究，这些酶在所有生命领域中负责复制和维持DNA。尽管经过仔细研究，关于聚合酶催化DNA链构建的机制以及限制其反应速度的因素仍有许多未知之处。荧光技术，特别是Förster共振能量转移（FRET），已经允许通过使用染料作为大分子的“分子尺子”来分析酶的结构。然而，这些方法无法观察到在30 Å以下的酶活性位点发生的较小变化。本项目将通过使用过渡金属阳离子配合物作为10-20 Å以上的FRET受体，将FRET扩展到研究酶的催化机制所需的规模，并检查DNA聚合酶活性位点发生的变化。拟议的研究和培训计划位于生物学（酶功能，遗传学）和物理学（光学，光物理学）的交叉点。申请人将获得物理科学方面的培训，特别是在单分子FRET的理论和应用、高水平光学仪器的使用和统计数据分析方面，建立申请人的生物学和遗传学背景。将FRET扩展到以前无法到达的距离，将广泛适用于研究大分子（生物和其他）中的小结构变化。更广泛地说，该项目通过将前沿研究与本科教育（课程开发、教学和学生指导）以及通过当地合作伙伴组织的公众宣传相结合，促进了其成果的广泛传播。

Understanding the Librarians of the Genome: developing new physical assays to detect conformational changes in DNA-processing enzymesDNA polymerases, enzymes responsible for replicating and maintaining DNA in all domains of life, have been extensively studied over the past half-century. Despite the scrutiny, much remains unknown about the mechanism by which polymerases catalyze the building of a DNA strand and what limits their rate of reaction. Fluorescence techniques, Förster Resonance Energy Transfer (FRET) in particular, have allowed analysis of enzyme structure by using dyes as 'molecular rulers' of macromolecules. However, these methods cannot observe the smaller changes below 30 Å that occur at the active sites of enzymes. This project will extend FRET to the scale needed to study catalytic mechanisms of enzymes through the use of transition metal cation complexes as FRET acceptors over 10-20 Å, and examine the changes that occur in the active site of DNA polymerases. The proposed research and training plan lies at the intersection of biology (enzyme function, genetics) and physics (optics, photophysics). The applicant will gain training in the physical sciences, particularly on the theory and application of single-molecule FRET, the use of high-level optical instrumentation, and statistical data analysis, building upon the applicant's biology and genetics background. The extension of FRET to previously-inaccessible distances will find broad applicability to studying small structural changes in large molecules -- biological and otherwise. More broadly, this project facilitates broad dissemination of its results by integrating cutting-edge research with undergraduate education (course development, teaching and student mentoring) and public outreach via local partner organizations.