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

有了这个奖项，化学部的生命过程化学计划和分子与细胞生物科学部的遗传机制计划正在资助来自加州大学欧文分校的John Chaput博士，以应用时间分辨X射线晶体学方法来捕获快照图像，因为DNA聚合酶将一条DNA链复制到另一条DNA链上。DNA是指导我们星球上生命的分子基础的蓝图。然而，尽管经过数十年的研究，人们仍然不完全了解酶如何在细胞内复制新的DNA。 这些图像的集合被组装起来，以产生相当于动画电影的效果，在原子水平上显示反应途径中每个步骤的精确顺序。对这些酶的化学机制的详细了解可能会导致新的聚合酶在生物技术中的应用设计。此外，该项目包括一个重要的教育部分，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计划，包括少数民族科学计划，旨在改善传统上代表性不足的群体在科学中的参与。 时间分辨X射线晶体学是一种强有力的方法，用于通过捕获在静态蛋白质晶体结构的最低能量状态中无法观察到的中间体来研究酶的机制。该项目通过收集嗜热脂肪芽孢杆菌（Bst）和水生栖热菌（KlenTaq）DNA聚合酶的Klenow片段类似物的时间推移图像来研究DNA合成的机制。我们的目标是提供细节的酪氨酸门控机制，防止移码突变和一种新的推拉机制在延长。此外，该研究还探索了一种由Bst聚合酶催化的不寻常的替代反应，该反应使用非规范模板（RNA，2-氟阿拉伯核酸或α-L-苏糖核酸）进行DNA延伸。的延时结构预计，以确定构象变化，因为它们发生在酶的活性位点。快照图像的完整收集为反应途径中每个中间体的精确顺序提供了明确的证据，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。该项目的成功完成有望阐明生命最基本过程之一-DNA合成的化学过程。该奖项由化学系生命过程化学项目共同资助（数学和物理科学（MPS）理事会）和遗传机制集群在分子和细胞生物科学司该奖项反映了NSF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.