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

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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.