NSF Postdoctoral Fellowship in Biology FY 2013

了解基因组的文库管理员：在过去的半个世纪里，人们对DNA加工酶--DNA聚合酶--负责复制和维持DNA的酶--的构象变化进行了广泛的研究。尽管进行了仔细的审查，但关于聚合酶催化DNA链构建的机制以及是什么限制了它们的反应速度，仍有许多未知之处。荧光技术，特别是Förster共振能量转移(FRET)，使人们能够通过将染料用作大分子的“分子尺子”来分析酶的结构。然而，这些方法不能观察到在酶的活性部位发生的30？以下的较小变化。该项目将通过使用过渡金属阳离子络合物作为FRET受体，将FRET扩展到研究酶的催化机制所需的规模，并研究DNA聚合酶活性部位发生的变化。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)的交叉点。申请者将获得自然科学方面的培训，特别是单分子FRET的理论和应用、高级光学仪器的使用和统计数据分析，以申请者的生物学和遗传学背景为基础。将FRET扩展到以前无法到达的距离，将发现它在研究大分子--生物和其他方面--的微小结构变化方面具有广泛的适用性。更广泛地说，该项目通过将尖端研究与本科教育(课程开发、教学和学生辅导)以及通过当地合作伙伴组织的公众宣传结合起来，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.