Structure and Mechanism of the Red beta Recombineering Enzyme

Red beta重组酶的结构和机制

• 批准号: 2212951
• 负责人: Charles Bell
• 金额: $ 104.14万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2212951&HistoricalAwards=false
• 关键词: Structure; Mechanism; Red; beta; Recombineering

This project will study a process by which bacteria can be re-engineered for useful purposes such as conversion of carbon dioxide into biofuels to mitigate climate change, remediation of toxic waste, and killing cancer cells. Bacterial re-engineering involves DNA recombination whereby new DNA sequences are added to bacterial cells and incorporated into their genomes by a protein called recombinase. The recombinase binds to the input DNA and pairs it with the host bacterial genome for incorporation during replication. In order to work efficiently, the recombinase must interact with single-stranded DNA binding protein (SSB), which is part of the bacterial replication machinery. This project will study how the recombinase binds to SSB, by visualizing the complex at atomic resolution and by measuring the strength and speed of the interactions. New knowledge about the interactions, and the corresponding ability to manipulate them, will help increase the efficiency of DNA recombination and expand the utility of genome engineering in different types of bacteria for new applications. The project will also train future scientists, with particular emphasis on high school students.The study focuses on the Red-beta protein from bacteriophage lambda that is part of a DNA recombination system used for replication and repair of DNA breaks. Red-beta catalyzes single-stranded DNA annealing (SSA), and its activity has been harnessed for powerful methods of bacterial genome engineering. In these methods Red-beta binds to transformed synthetic DNA and anneals it to the existing bacterial genome as it is undergoing replication. This process requires interaction between Red-beta and SSB protein, which binds and protects the lagging strand of the replication fork. This project will use x-ray crystallography and cryo-electron microscopy to determine three-dimensional structures of the Red-beta-SSB complex, and other biophysical methods to determine the energetics of the binding interaction. The resulting knowledge will be used to fine-tune the Red-beta-SSB interaction and to make it compatible in a wider range of bacterial hosts. The project will also provide laboratory research training to students at all levels, with particular emphasis on high school students.This project is jointly funded by the Genetic Mechanisms and Molecular Biophysics programs of the Molecular and Cellular Biosciences Division in the Biological Sciences 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.

该项目将研究一个可以重新设计细菌以进行有用目的的过程，例如将二氧化碳转化为生物燃料，以减轻气候变化，修复有毒废物和杀死癌细胞。细菌重新设计涉及DNA重组，从而将新的DNA序列添加到细菌细胞中，并通过称为重组酶的蛋白质掺入其基因组中。重组酶与输入DNA结合，并将其与宿主细菌基因组配对以在复制过程中掺入。为了有效工作，重组酶必须与单链DNA结合蛋白（SSB）相互作用，这是细菌复制机制的一部分。该项目将通过在原子分辨率下观察复合物以及测量相互作用的强度和速度来研究重组酶如何与SSB结合。有关相互作用的新知识以及对它们进行操作的相应能力，将有助于提高DNA重组的效率，并扩大不同类型的细菌中基因组工程的实用性。该项目还将培训未来的科学家，特别着重于高中生。该研究的重点是噬菌体lambda的红β蛋白，该蛋白是用于复制和修复DNA断裂的DNA重组系统的一部分。红β催化了单链DNA退火（SSA），其活性已被利用，用于有力的细菌基因组工程方法。在这些方法中，红β与转化的合成DNA结合，并在复制时将其退火到现有的细菌基因组。此过程需要红β和SSB蛋白之间的相互作用，该蛋白结合并保护复制叉的滞后链。该项目将使用X射线晶体学和冷冻电子显微镜来确定红β-SSB复合物的三维结构，以及其他生物物理方法来确定结合相互作用的能量。所得的知识将用于微调红色β-SSB相互作用，并使其在更广泛的细菌宿主中兼容。该项目还将向各个级别的学生提供实验室研究培训，特别着重于高中生。该项目由生物科学局的分子和蜂窝生物科学局的遗传机制和分子生物物理学计划共同资助。该奖项是NSF的法定任务，反映出值得及其构成的支持者，该奖项反映了综述的依据，这是由Infestia and Intelliquia的构成及其构成的作用。

项目成果

Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing.

• DOI: 10.1038/s41467-022-35572-z
• 发表时间: 2022-12-21
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Caldwell, Brian J.;Norris, Andrew S.;Karbowski, Caroline F.;Wiegand, Alyssa M.;Wysocki, Vicki H.;Bell, Charles E.
• 通讯作者: Bell, Charles E.