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

通过时间分辨 X 射线晶体学阐明复制 DNA 合成的隐藏步骤

基本信息

批准号：
2001434
负责人：
John Chaput
金额：
$ 43.8万
依托单位：
University of California-Irvine
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2001434&HistoricalAwards=false
关键词：
Elucidating Hidden Steps Replicative DNA

项目摘要

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.

获得这一奖项后，化学部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John Chaput博士应用时间分辨X射线结晶学方法，在DNA聚合酶将一条DNA复制到另一条链时捕捉快照图像。DNA是指导我们星球上生命的分子基础的蓝图。然而，尽管进行了数十年的研究，人们对酶如何在细胞内制造新的DNA副本仍有不完全的理解。图像集合被组合在一起，以产生相当于动画电影的效果，在原子水平上显示反应路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新的聚合酶应用于生物技术。此外，该项目还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由大学赞助的项目，包括旨在改善传统上代表性不足的群体参与科学的少数群体科学计划。时间分辨X射线结晶学是通过捕捉在静态蛋白质晶体结构的最低能态下观察不到的中间体来研究酶的机制的有力方法。本项目通过采集嗜热脂肪芽孢杆菌(BST)和水热杆菌(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。其目的是提供防止帧移位突变的酪氨酸门控机制的细节，以及在伸长过程中的新的推拉机制。此外，这项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(核糖核酸、2-氟-阿拉伯核酸或α-L-苏糖核酸)来延长DNA。当它们发生在酶活性部位时，时间推移结构被投射来识别构象变化。完整的快照图像收集是为了提供明确的证据，证明每个中间体在反应途径中的准确顺序，并可能导致发现以前的DNA聚合酶研究中没有观察到的新的中间体。这个项目的成功完成有望阐明生命中最基本的过程之一-DNA合成的化学。该奖项由化学部(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(生物科学(BIO)理事会)的遗传机制群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。