Structural Biology of DNA Repair by Single-Strand Annealing

单链退火修复 DNA 的结构生物学

• 批准号: 1021966
• 负责人: Charles Bell
• 金额: $ 53.25万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1021966&HistoricalAwards=false
• 关键词: Structural; Biology; DNA; Repair; Single

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.

智力优势：所有有机体的细胞不断地暴露在环境侮辱中，破坏它们的DNA及其编码的遗传信息。双链DNA(DsDNA)断裂尤其有害，即双链的两条链在同一位置断裂。幸运的是，细胞有许多修复dsDNA断裂的方法。一种重要的修复途径被称为“单链退火”(SSA)，它涉及切除(向后修剪)暴露在断裂处的DNA末端，形成两个单链DNA悬垂，然后相互退火以修复断裂。SSA由真核生物中包括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”)重组系统启动。Recet和Redab重组系统都由两个蛋白质组成：一个是高度进行性的5‘-3’外切酶Ress或Reda，它与dsDNA末端结合并消化5‘端的链，另一个是单链退火蛋白RECT或RedB，它与产生的3’-突出物结合，以促进单链DNA的互补链(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个被称为“突触体”的复合体，当它由核酸外切酶产生时，它可能用于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，为理解SSA的基本机制原理提供了一个方便的模型。此外，由于他们能够在短的同源性区域工作，Recet和Redab最近被部署来创造被称为“重组工程”的强大的遗传工程新方法。核酸外切酶还被用于单分子纳米孔DNA测序的新方法中。尽管RECET和REDAB作为模型系统很重要，并且它们出现在强大的新生物技术应用中，但在机制水平上对这些蛋白质的理解还不是很好，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，将测定Ress和Reda与DNA底物形成的络合物的X射线晶体结构，以揭示它们是如何与dsDNA末端结合并连续消化DNA底物的。在目标2中，将确定RECT和REDB的晶体结构，以揭示它们是如何与单链DNA结合并促进互补链的退火的。这些研究将为理解SSA蛋白的潜在机制提供基础。从这些研究中获得的知识也将为设计性能增强的新蛋白质应用于基因工程和纳米孔DNA测序铺平道路。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。研究生将从俄亥俄州立大学现有的项目中招聘，包括俄亥俄州立大学生物化学计划(OSBP)、生物物理学研究生计划和化学-生物接口计划。PI还将通过俄亥俄州立大学的暑期研究机会计划(SROP)为来自未被充分代表的少数族裔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 High School建立了合作伙伴关系，招募高中生参加带薪暑期实习。麦德龙是一所新成立的、专注于STEM的高中，位于俄亥俄州立大学附近，为来自低收入城市社区的学生提供参与高级大学早期科学课程的机会。麦德龙大学的学生通常在初中时开始在俄亥俄州立大学上课，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每年将招聘一名麦德龙学生到实验室工作，作为带薪暑期实习生。学生将有机会参与X射线结构测定的方方面面，包括目标蛋白质的克隆、表达和结晶，以及X射线结构测定和分析。PI还将通过他在Biomedical Partnership Team的服务参加Metro High School的活动，Biomedical Partnership Team是一个由当地科学家组成的委员会，帮助为高级地区的高中生设计创新的科学课程。