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.
Intellectual Merit. By using synthetic RNA-containing oligonucleotides, we characterized the process how RNA can repair DNA damage in yeast Saccharomyces cerevisiae cells and explored this activity also in the bacterium Escherichia coli and human embryonic kidney (HEK-293) cells. We studied the capacity of DNA repair factors to target RNA/DNA substrates in S. cerevisiae and E. coli. We found that the mismatch repair (MMR) system, which is known to repair almost any kind of DNA/DNA mispairs can also target mispaired ribonucleotides (rNMPs) in DNA in yeast and E. coli. Our results uncovered different mechanisms by which the amount of rNMPs in DNA may be regulated either to prevent alterations of DNA, or perhaps to modulate gene function. In a collaborative effort, we found that rNMP intrusions in DNA are able to induce local structural distortions involving the rNMP and the nucleotide 3’ from it. These distortions alter the torsional angles alpha and gamma of the sugar-phosphate backbone and decrease – by 32% – the stretch modulus of DNA depending on specific flanking sequences. These results derive from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamic simulations of rNMP-containing DNA, and from experiments of atomic force microscopy (AFM) using rNMP-containing single molecules. Because rNMPs are the most abundant non-standard nucleotides that can be found in DNA of cells, with the goal to further understand the function and consequences of rNMPs in DNA, we invented and developed an approach to capture and map rNMPs incorporated in genomic DNA. We then utilized our approach for rNMP capture to determine the spectrum of rNMP incorporation in the nuclear and mitochondrial genome of S. cerevisiae, and we found non-random distribution with particular hotspots. With the objective to determine whether transcript RNA can play an active role in DNA repair, we set up a system in yeast to examine the capacity of transcript RNA to directly or indirectly serve as a template for chromosomal DNA double-strand break (DSB) repair via homologous recombination. This work led to the discovery of transcript RNA-mediated repair of a DNA DSB in yeast. We showed that the transfer of genetic information from RNA to DNA occurs with an endogenous generic transcript, and is thus a more general phenomenon than previously anticipated. In addition, we found that in vitro RNA–DNA annealing is markedly promoted not only by the yeast recombination protein Rad52 but also by the human RAD52 enzyme, suggesting that transcript-RNA-templated DNA repair could occur in human cells. Our findings reveal the presence of a novel mechanism of DNA repair and homologous recombination mediated by RNA. These studies resulted in 8 published, 1 accepted, 1 in review and 1 submitted manuscripts. Publications include journals such as Nature Structural & Molecular Biology, Nanoscale, and Nature. Broader Impacts. Results obtained with NSF support have been used in teaching efforts in all courses taught by the PI for undergraduates, graduates, or both, and served for training and mentoring 9 undergraduate and 7 graduate students, 6 high school students and a research scientist. Four undergraduates are authors in publications resulted from this NSF grant project. Two of these undergraduates share first authorship with a graduate student, and one of these undergraduates is author in the manuscript published in Nature. Five of the 9 trained undergraduates went to medical schools and 4 are applying or already are in a graduate program. Five undergraduates won the President’s Undergraduate Research Award (PURA) and/or other awards while working in the PI’s lab. The two graduate students who were fully supported by this NSF grant have each three first-author published/accepted or in review manuscripts, and at least one second-author publication, up to now, all on the work of this NSF project. Other 5 graduate students were also involved in the project to different extent, and each, except 1 who just recently joined the lab, have been an author, including first author, in one or more publications from this study. The graduate students (3), who were fully involved in this NSF project, each won from 1 to 3 awards at conferences and made numerous oral and poster presentations of their work at national/international conferences as well as local institutions. The research scientist provided technical assistance and relevant contribution for the NSF grant. The high school students (6) assisted the work of the graduate students and learned various basic laboratory techniques. The PI was invited to give talks on the findings of this NSF project at 12 conferences and at 8 national/international institutions. A couple of these talks also included audience from the general public. Moreover, the PI and a colleague hosted a visit to their labs of a group of people from the general public. The visit received excellent feedback and was also a terrific experience for the graduate students in the PI’s lab.
