Structural Biology of DNA Repair by Single-Strand Annealing

智力优点：所有生物体的细胞都不断暴露在环境的侮辱中，破坏了它们的DNA和它编码的遗传信息。双链DNA（dsDNA）断裂，其中双链体的两条链在相同位置断裂，是特别有害的。幸运的是，细胞有许多修复dsDNA断裂的方法。一个重要的修复途径，称为“单链退火”（SSA），涉及切除（修剪）在断裂处暴露的DNA末端以形成两个单链DNA突出端，然后使其彼此退火以修复断裂。SSA由真核生物中包括Rad 52的蛋白质网络或细菌中相对简单的噬菌体编码的RecET和Redab（Red“alpha/beta”）重组系统促进。RecET和Redab重组系统各自由两种蛋白质组成：高度进行性的5 '-3'核酸外切酶RecE或Reda，其结合到dsDNA末端并使5 '末端链突出;以及单链退火蛋白RecT或Redb，其结合到所得的3'突出端以促进其与单链DNA（ssDNA）的互补链退火。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”的复合物，当单链退火蛋白由核酸外切酶产生时，该复合物可以将单链退火蛋白加载到3 '-突出端上。RecET和Redab重组系统是高度进化和高效的，并为理解SSA的基本机制原理提供了一个方便的模型。此外，由于RecET和Redab能够在短同源区域工作，它们最近被用于创建称为“重组工程”的强大的基因工程新方法。“核酸外切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，但蛋白质在机制水平上还没有得到很好的理解，这在很大程度上是由于缺乏结构信息。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。在目标1中，将确定与DNA底物复合的RecE和Reda的X射线晶体结构，以揭示它们如何结合到dsDNA末端和Proclase消化DNA底物。在目标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新的蛋白质铺平道路，这些蛋白质具有增强的性质，可用于基因工程和纳米孔DNA测序。更广泛的影响本项目将为研究生、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的既定项目中招募，包括俄亥俄州生物化学项目（OSBP），生物物理研究生项目和化学生物学接口项目。PI还将通过OSU的夏季研究机会计划（SROP）为来自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的地铁高中建立了合作伙伴关系，招募高中生进行带薪暑期实习。Metro是一所新成立的，以STEM为重点的高中，位于OSU附近，为来自低收入城市社区的学生提供了参加先进的，早期大学的，以科学为基础的课程的机会。地铁学生通常开始在他们的大三在俄勒冈州立大学上课，并鼓励参加动手研究活动，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还将通过他在生物医学伙伴关系小组的服务参加地铁高中的活动，该小组是一个由当地科学家组成的委员会，帮助为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.