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Evolved DNA contacts required for hexameric helicase unwinding

六聚体解旋酶解旋所需的进化DNA接触

基本信息

批准号：
1613534
负责人：
Michael Trakselis
金额：
$ 78万
依托单位：
Baylor University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613534&HistoricalAwards=false
关键词：
Evolved DNA contacts required hexameric

项目摘要

Hexameric DNA replication helicases are structurally conserved and essential enzymes present throughout all domains of life including most viruses. They utilize a common topological strategy of encircling one strand of duplex DNA to physically separate the duplex by coupled ATP hydrolysis, which results in separated single-strand DNA templates for leading and lagging strand DNA synthesis. Earlier research in the field has focused on DNA contacts within the central channel of these helicases, while recently identified exterior contacts with the excluded DNA strand have largely remained unexplored. This project will assess the importance of external helicase contacts in controlling the speed of DNA replication through direct or indirect interactions with the excluded strand. Because a multitude of hexameric helicases across all domains of life will be compared for the nature of the interactions and the consequences of disrupting them, both in vitro and in vivo, the scientific scope and impact is expected to be broad and influential. Undergraduate and graduate students will be trained in techniques of single molecule fluorescence, advanced enzyme kinetics and precise genetic manipulation. Scientific outreach programs will encourage local elementary school students to explore the wonders of their own genome through "DNA Days" and engage students in electromagnetism by building their own working audio speakers. The mechanism of DNA unwinding by hexameric helicases during genome replication currently focuses solely on interactions with the encircled strand and overlooks the implication of contacts with the excluded strand. Preliminary work suggests that these external contacts could be more influential in controlling the speed of replication, coupling enzymes at the replication fork and sensing DNA damage. This project will interrogate and quantify precise chemical specificities of excluded strand interactions, and monitor mutational consequences on replisome speed, enzymatic coordination, and genomic stability, both in vitro and in vivo. The resulting data, methods, and findings will be broadly distributed within the scientific community and will provide further insights and comparisons into the precise mechanisms of action of hexameric helicases in DNA replication across all domains of life.This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate for Mathematical and Physical Sciences.

六聚体 DNA 复制解旋酶在结构上是保守的，是生命所有领域（包括大多数病毒）中必需的酶。他们利用一种常见的拓扑策略，即环绕双链体 DNA 的一条链，通过耦合 ATP 水解来物理分离双链体，从而产生用于前导链和滞后链 DNA 合成的分离的单链 DNA 模板。该领域的早期研究主要集中在这些解旋酶中央通道内的 DNA 接触，而最近发现的与排除的 DNA 链的外部接触在很大程度上仍未被探索。该项目将评估外部解旋酶接触在通过与排除链直接或间接相互作用来控制 DNA 复制速度方面的重要性。由于将在体外和体内比较生命各个领域的大量六聚体解旋酶的相互作用的性质以及破坏它们的后果，因此科学范围和影响预计将是广泛且有影响力的。本科生和研究生将接受单分子荧光、先进酶动力学和精确基因操作技术的培训。科学推广计划将鼓励当地小学生通过“DNA Days”探索自己基因组的奇迹，并通过制作自己的工作音频扬声器让学生接触电磁学。目前，基因组复制过程中六聚体解旋酶解旋 DNA 的机制仅关注与环绕链的相互作用，而忽略了与排除链接触的影响。初步研究表明，这些外部接触可能在控制复制速度、复制叉处的酶偶联和感知 DNA 损伤方面更具影响力。该项目将询问和量化排除链相互作用的精确化学特异性，并在体外和体内监测对复制体速度、酶协调和基因组稳定性的突变后果。由此产生的数据、方法和发现将在科学界广泛传播，并将为生命各个领域的 DNA 复制中六聚体解旋酶的精确作用机制提供进一步的见解和比较。该项目由生物科学局分子和细胞生物科学部遗传机制集群和生物科学部生命过程化学项目联合资助。数学和物理科学理事会的化学。