Structural Biology of DNA Repair by Single-Strand Annealing

智力价值：所有生物体的细胞都不断暴露于环境的损害中，这会损害它们的DNA及其编码的遗传信息。双链DNA （dsDNA）断裂，双链的两条链在同一位置断裂，是特别有害的。幸运的是，细胞有许多修复双链dna断裂的方法。一种重要的修复途径，称为“单链退火”（SSA），包括切除（修剪）断裂处暴露的DNA末端，形成两个单链DNA悬垂，然后彼此退火以修复断裂。真核生物中包括Rad52在内的蛋白质网络或细菌中相对简单的噬菌体编码的RecET和Redab（红色“α / β”）重组系统可促进SSA。RecET和Redab重组系统分别由两种蛋白质组成：一种是高进程性的5‘-3’外切酶RecE或Reda，它结合到dsDNA末端并消化5‘末端的链，另一种是单链退火蛋白RecT或Redb，它结合到产生的3’-悬垂，促进其与互补的单链DNA （ssDNA）的退火。有趣的是，每个系统的两种蛋白质相互结合，形成一种称为“突触体”的复合体，当它由外切酶产生时，它可能用于将单链退火蛋白加载到3'-悬垂上。RecET和Redab重组系统是高度进化和高效的，为理解SSA的基本机制原理提供了一个方便的模型。此外，由于它们能够在短同源区域工作，RecET和Redab最近被用于创建强大的基因工程新方法，称为“重组”。外切酶酶也被用于单分子纳米孔DNA测序的新方法中。尽管RecET和Redab作为模型系统的重要性，以及它们在强大的新生物技术应用中的出现，但由于缺乏结构信息，这些蛋白质在机制水平上并没有得到很好的理解。该项目的长期目标是应用结构生物学、生物化学和遗传学的工具，在原子水平上阐明RecET和Redab重组系统的内部工作原理。在Aim 1中，将确定RecE和Reda与DNA底物复合物的x射线晶体结构，以揭示它们如何与dsDNA末端结合并逐步消化DNA底物。在Aim 2中，将确定RecT和Redb的晶体结构，以揭示它们如何与ssDNA结合并促进互补链的退火。这些研究将为理解SSA蛋白的潜在机制原理提供基础。从这些研究中获得的知识也将为设计具有增强性能的新蛋白质铺平道路，用于基因工程和纳米孔DNA测序。更广泛的影响该项目将为研究生、本科生和高中生的培训提供丰富的机会。研究生将从俄勒冈州立大学已建立的项目中招募，包括俄亥俄州立生物化学项目（OSBP）、生物物理学研究生项目和化学-生物界面项目。该项目还将通过俄勒冈州立大学的暑期研究机会计划（SROP）为来自代表性不足的少数民族背景的本科生提供研究和培训机会。此外，PI还与俄亥俄州哥伦布市的Metro高中建立了合作关系，招收高中生进行带薪暑期实习。Metro是俄勒冈州立大学附近一所新成立的以stem为重点的高中，它为来自低收入城市社区的学生提供了参加高级、早期大学科学课程的机会。Metro学生通常在他们的大三开始在OSU上课，并被鼓励参加实践研究活动，以加强他们的课堂学习。为此，PI每年将招募一名Metro学生作为带薪暑期实习生在实验室工作。学生将有机会参与x射线结构测定的各个方面，包括靶蛋白的克隆、表达和结晶，以及x射线结构测定和分析。PI还将通过生物医学合作小组（Biomedical Partnership Team）的服务，参与Metro高中的活动。生物医学合作小组是一个由当地科学家组成的委员会，旨在帮助先进地区的高中学生设计创新的科学课程。

Intellectual Merit:The cells of all organisms are exposed constantly to environmental insults that damage their DNA and the genetic information it encodes. Double stranded DNA (dsDNA) breaks, in which both strands of the duplex are broken at the same position, are particularly harmful. Fortunately, cells have many ways of repairing dsDNA breaks. An important repair pathway, called "single-strand annealing" (SSA), involves resection (trimming back) of the DNA ends exposed at the break to form two single-stranded DNA overhangs, which are then annealed to one another to repair the break. SSA is promoted by a network of proteins including Rad52 in eukaryotes, or by the relatively simple phage-encoded RecET and Redab (Red "alpha/beta") recombination systems in bacteria. The RecET and Redab recombination systems each consist of two proteins: a highly processive 5'-3' exonuclease, RecE or Reda, which binds to dsDNA ends and digests the 5'-ended strand, and a single-strand annealing protein, RecT or Redb, which binds to the resulting 3'-overhang to promote its annealing with a complementary strand of single-stranded DNA (ssDNA). Interestingly, the two proteins of each system bind to one another to form a complex known as a "synaptasome," which may serve to load the single-strand annealing protein onto the 3'-overhang as it is generated by the exonuclease. The RecET and Redab recombination systems are highly evolved and efficient, and offer a convenient model for understanding the basic mechanistic principles of SSA. Moreover, due to their ability to work at short regions of homology, RecET and Redab have recently been deployed to create powerful new methods for genetic engineering called "recombineering." The exonuclease enzymes are also being exploited in new methods for single-molecule nanopore DNA sequencing. In spite of the importance of RecET and Redab as model systems, and their emergence in powerful new biotechnology applications, the proteins are not well understood at the mechanistic level, in large part due to a lack of structural information. The long-term goals of this project are to apply the tools of structural biology, biochemistry and genetics to elucidate the inner workings of the RecET and Redab recombination systems at the atomic level. In Aim 1, x-ray crystal structures of RecE and Reda in complex with DNA substrates will be determined, to reveal how they bind to dsDNA ends and processively digest DNA substrates. In Aim 2, crystal structures of RecT and Redb will be determined, to reveal how they bind to ssDNA and promote the annealing of complementary strands. These studies will provide a foundation for understanding the underlying mechanistic principles of SSA proteins. The knowledge gained from these studies will also pave the way for the design of new proteins with enhanced properties for applications in genetic engineering and nanopore DNA sequencing.Broader ImpactsThis project will provide rich opportunities for the training of graduate, undergraduate, and high school students. Graduate students will be recruited from established programs at OSU, including the Ohio State Biochemistry Program (OSBP), the Biophysics Graduate Program, and the Chemistry-Biology Interface Program. The PI will also provide research and training opportunities to undergraduate students from under-represented minority backgrounds, recruiting through OSU's Summer Research Opportunities Program (SROP). In addition, the PI has a partnership with Metro High School in Columbus, Ohio to recruit high school students for paid summer internships. Metro is a newly formed, STEM-focused high school near OSU that gives students from low-income, urban neighborhoods the opportunity to participate in an advanced, early college, science-based curriculum. Metro students typically begin taking classes at OSU during their Junior years, and are encouraged to participate in hands-on research activities that reinforce their classroom studies. Towards this end, the PI will recruit one Metro student each year to work in the laboratory as a paid summer intern. The students will be given the opportunity to participate in all aspects of x-ray structure determination, including cloning, expression, and crystallization of target proteins, as well as x-ray structure determination and analysis. The PI will also participate in activities at Metro High School through his service on the Biomedical Partnership Team, a committee of local scientists that helps to design innovative science curricula for advanced area high school students.