Mechanisms of RNA/DNA Hybrid Stability and of Information Flow From RNA to DNA in Yeast Cells

知识价值：在分子生物学的中心法则中，信息通常从DNA流向RNA，再从RNA流向蛋白质。只有在逆转录病毒、逆转录转座子和端粒合成等特殊情况下，RNA信息才能通过逆转录复制到DNA中。尽管在过去的几十年里，人们发现了RNA惊人的多种功能，但一直很难确定RNA是否能直接修饰细胞的基因组。利用合成的含有RNA的寡核苷酸（oligos），最近的研究表明，RNA可以作为修复基因组位点特异性双链断裂（DSB）的模板，并可以将遗传信息传递到酵母（Saccharomyces cerevisiae）的染色体DNA上。值得注意的是，一些出版物支持核糖核苷酸在DNA合成过程中经常被纳入细胞基因组的可能性。然而，目前尚不清楚这种RNA/DNA杂交的稳定性，以及这些杂交在多大程度上影响细胞的遗传完整性。这项研究的目的是了解RNA直接将信息传递给细胞DNA的机制。重点将集中在以下目标上：1)确定在RNA驱动的DNA修复和DNA修饰过程中，RNA/DNA杂交体中切割RNA通道的主要蛋白质因子，并表征它们的体内功能。2)发现和定义DNA修复机制在去除嵌入DNA的RNA中的作用。3)鉴定RNA驱动DNA修复和RNA嵌入基因组DNA时，主要通过RNA束参与DNA合成的DNA聚合酶/s。该项目的结果将提高我们对基因组稳定性基本机制的认识，并将阐明RNA在DNA编辑和重塑中发挥积极作用的能力，这可能是RNA驱动的DNA进化完全未开发过程的基础。这项研究还可以为开发新的基因靶向/治疗方法提供重要的见解，这些方法可以利用RNA修饰基因组DNA的能力。更广泛的影响：研究在很大程度上依赖于本科生和研究生的积极参与，他们将被训练学习科学思维和实验室技术，并准备在未来成为好奇和批判性的科学家。所得结果将作为学士、硕士、博士学位论文答辩的依据。在RNA生物学和DNA修复方面的新发现将被整合到PI教授的课程中。此外，项目支持将有助于增加吸引高中生开始实验室经验的机会，并将鼓励他们从事科学事业。研究团队的多样性将通过招募女性研究人员和少数群体成员来实现，这些成员占佐治亚理工学院学生人数的很大一部分。

Intellectual Merit: In the central dogma of molecular biology, information generally flows from DNA to RNA and from RNA to proteins. RNA information can be copied into DNA via reverse transcription only in the special cases of retroviruses, retrotransposons and telomere synthesis. Although an astonishing variety of RNA functions have been found in the last few decades, it has always been very difficult to determine if RNA can directly modify the genome of cells. Exploiting the use of synthetic RNA-containing oligonucleotides (oligos), it was recently shown that RNA can serve as a template for the repair of a genomic site-specific double-strand break (DSB) and can transfer genetic information to chromosomal DNA in the yeast, Saccharomyces cerevisiae. Notably, several publications support the possibility that ribonucleotides can frequently be incorporated into the genome of cells during DNA synthesis. However, it is not known how stable such RNA/DNA hybrids can be and to what extent these hybrids can affect the genetic integrity of cells. The goal of this research is to understand the mechanisms by which RNA can directly transfer information to the DNA of cells. Focus will be on the following objectives: 1) To identify the main protein factors cleaving the RNA tract in an RNA/DNA hybrid during RNA-driven DNA repair and DNA modification, and to characterize their in vivo function. 2) To discover and define the role of DNA repair mechanisms in the removal of RNA embedded into DNA. 3) To identify the DNA polymerase/s mainly participating in DNA synthesis through RNA tracts during RNA-driven DNA repair and when RNA is embedded into genomic DNA. Results from this project will improve our knowledge on the basic mechanisms of genome in/stability and will shed light on the capacity of RNA to play an active role in DNA editing and remodeling, which could be the basis of a wholly unexplored process of RNA-driven DNA evolution. This research could also provide important insights for the development of novel gene targeting/therapy approaches that could exploit the capacity of RNA to modify genomic DNA. Broader impacts: The research heavily relies on the active participation of undergraduate and graduate students, who will be trained to learn scientific thinking and laboratory techniques and will be prepared to become inquisitive and critical scientists in the future. The results obtained in this work will be used as a basis for the defense of B.S., M.S. and Ph.D. theses. The new findings in RNA biology and DNA repair will be integrated in the lecture program of the courses taught by the PI. Moreover, project support will serve to enhance opportunities to attract high school students to initiate a laboratory experience and will encourage them to pursue careers in the sciences. The diversity of the research team will be achieved by recruiting female researchers and members of minority groups that constitute significant fraction of student population at Georgia Institute of Technology.