Elucidating the Hidden Steps of Replicative DNA Synthesis by Time-Resolved X-ray Crystallography

有了这个奖项，化学部门的生命过程化学项目和分子和细胞生物科学部的遗传机制项目将资助加州大学欧文分校的约翰·查普特博士，应用时间分辨x射线晶体学方法来捕捉DNA聚合酶将一条DNA链复制到另一条DNA链上的快照图像。DNA是指导地球上生命分子基础的蓝图。然而，尽管经过了几十年的研究，人们对酶如何在细胞内复制新的DNA仍然知之甚少。这些图像集合在一起，就像一部动画电影，展示了原子水平上反应途径中每一步的精确顺序。对这些酶的化学机制的详细了解可能会导致设计用于生物技术的新聚合酶。此外，该项目包括一个重要的教育组成部分，旨在吸引和保持学生对化学和生物科学的兴趣。这包括积极参与一些大学赞助的项目，包括少数民族科学项目，旨在提高传统上代表性不足的群体对科学的参与。时间分辨x射线晶体学是研究酶的机制的一种强有力的方法，它可以捕获在静态蛋白质晶体结构的最低能态中无法观察到的中间体。本项目通过收集嗜热脂肪芽孢杆菌（Bst）和水生热杆菌（KlenTaq） DNA聚合酶的klenow片段类似物的延时图像来研究DNA合成的机制。目的是提供酪氨酸门控机制的细节，防止帧移位突变和一种新的推拉机制在伸长。此外，该研究还探讨了由Bst聚合酶催化的一种不寻常的替代反应，该反应使用非规范模板（RNA， 2-氟阿拉伯核酸或α - l -蔗糖核酸）进行DNA延伸。随着时间推移的结构被预测，以确定构象的变化，因为他们发生在酶的活性部位。完整的快照图像收集是为了为反应途径中每个中间体的精确顺序提供明确的证据，并可能导致发现以前DNA聚合酶研究中未观察到的新中间体。这个项目的成功完成有望阐明生命中最重要的过程之一——dna合成的化学过程。该奖项由化学部门（数学和物理科学（MPS）理事会）的生命过程化学项目和分子和细胞生物科学部门（生物科学（BIO）理事会）的遗传机制集群共同资助。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。

With this award, the Chemistry of Life Processes program in the Chemistry Division and the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences are funding Dr. John Chaput from the University of California, Irvine to apply time-resolved X-ray crystallography methods to capture snapshot images as the DNA polymerase enzyme copies one strand of DNA onto another. DNA is the blueprint that directs the molecular basis of life on our planet. However, despite decades of research, there remains an incomplete understanding of how enzymes make new copies of DNA inside cells. The collection of images is assembled to produce the equivalent of an animated movie showing the precise order of each step in the reaction pathway at the atomic level. The detailed insights into the chemical mechanism of these enzymes leads potentially to the design of new polymerases for applications in biotechnology. In addition, this project includes a significant educational component that is designed to attract and maintain student interest in the chemical and biological sciences. This includes pro-active engagement in a number of university-sponsored programs, including the Minority Science Program, aimed at improving the participation of traditionally underrepresented groups in science. Time-resolved X-ray crystallography is a powerful method for studying the mechanism of enzymes by capturing intermediates that cannot be observed in the lowest energy states of static protein crystal structures. This project investigates the mechanism of DNA synthesis by collecting time-lapsed images of the Bacillus stearothermophilus (Bst) and the Klenow-fragment analogue of the Thermus aquaticus (KlenTaq) DNA polymerase. The objectives are to provide details of a tyrosine gating mechanism that prevents frame-shift mutations and a novel push-pull mechanism during elongation. In addition, the study probes an unusual alternative reaction catalyzed by Bst polymerase that uses non-canonical templates (RNA, 2-fluoro-arabino nucleic acid, or alpha-L-threose nucleic acid) for DNA elongation. The time-lapse structures are projected to identify conformational changes as they occur in the enzyme active site. The full collection of snapshot images is to provide unequivocal evidence for the precise order of each intermediate in the reaction pathway and may lead to the discovery of new intermediates not observed in previous DNA polymerase studies. Successful completion of this project is expected to illuminate the chemistry of one of life’s most essential processes—DNA synthesis.This award is co-funded by the Chemistry of Life Processes program in the Division of Chemistry (Mathematical and Physical Sciences (MPS) Directorate) and the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences (Biological Sciences (BIO)Directorate).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.