Structure and Mechanism of the Red beta Recombineering Enzyme

这个项目将研究一个过程，通过这个过程，细菌可以被重新设计，用于有用的目的，比如将二氧化碳转化为生物燃料，以缓解气候变化，修复有毒废物，杀死癌细胞。细菌重组包括DNA重组，即新的DNA序列被添加到细菌细胞中，并通过一种称为重组酶的蛋白质整合到它们的基因组中。重组酶与输入DNA结合，并与宿主细菌基因组配对，以便在复制过程中整合。为了有效地工作，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。该项目将研究重组酶如何与SSB结合，方法是在原子分辨率下观察复合体，并测量相互作用的强度和速度。关于相互作用的新知识，以及相应的操纵它们的能力，将有助于提高DNA重组的效率，并扩大基因组工程在不同类型细菌中的新应用。该项目还将培养未来的科学家，尤其是高中生。这项研究的重点是来自噬菌体的红β蛋白，它是DNA重组系统的一部分，用于复制和修复DNA断裂。红β催化单链DNA退火（SSA），其活性已被利用为细菌基因组工程的有力方法。在这些方法中，red - β与转化的合成DNA结合，并在现有细菌基因组进行复制时将其退火。这个过程需要红β和SSB蛋白之间的相互作用，SSB蛋白结合并保护复制叉的滞后链。本项目将使用x射线晶体学和低温电子显微镜来确定红- β - ssb复合物的三维结构，并使用其他生物物理方法来确定结合相互作用的能量学。由此产生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。该项目还将为各级学生提供实验室研究培训，重点是高中生。该项目由生物科学理事会分子和细胞生物科学部的遗传机制和分子生物物理学项目共同资助。该奖项反映了美国国家科学基金会的法定使命，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。

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.