Evolved DNA contacts required for hexameric helicase unwinding

六聚体DNA复制解旋酶在结构上是保守的，并且存在于包括大多数病毒在内的所有生命领域中的必需酶。它们利用环绕双链体DNA的一条链的共同拓扑策略，通过偶联ATP水解物理分离双链体，这导致分离的单链DNA模板用于前导和滞后链DNA合成。该领域的早期研究主要集中在这些解旋酶的中央通道内的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。本项目将评估外部解旋酶接触在通过与被排除链的直接或间接相互作用控制DNA复制速度方面的重要性。由于大量的六聚体解旋酶在生命的所有领域将比较的相互作用的性质和破坏它们的后果，在体外和体内，科学的范围和影响，预计将是广泛的和有影响力的。本科生和研究生将接受单分子荧光，先进酶动力学和精确遗传操作技术的培训。科学推广计划将鼓励当地小学生通过“DNA日”探索自己基因组的奇迹，并通过建造自己的工作音频扬声器让学生参与电磁学。 在基因组复制过程中通过六聚体解旋酶的DNA解旋机制目前仅关注与被包围的链的相互作用，而忽略了与被排除的链接触的含义。初步研究表明，这些外部接触可能在控制复制速度、在复制叉处偶联酶和感知DNA损伤方面更有影响力。该项目将询问和定量排除链相互作用的精确化学特异性，并监测体外和体内复制体速度，酶协调和基因组稳定性的突变后果。由此产生的数据，方法，该项目由生物科学理事会分子和细胞生物科学部遗传机制小组和生命过程化学计划共同资助，将在科学界广泛传播，并将为生命所有领域的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。数学和物理科学理事会化学部。

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.