Discovery, characterization and mechanism of RNA and CDNA-mediated DNA double-strand break repair

A double-strand break (DSB) is one of the most deleterious DNA lesions and its repair is crucial for genome stability. Even if a single DSB is not repaired precisely, this could cause mutations, chromosomal rearrangements, cell death, and apoptosis. The safest mechanism to repair a DSB is homologous recombination (HR). HR requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome to retrieve the missing genetic information and accomplish error-free repair. In special cases, HR can occur between RNA molecules, such as RNA molecules in RNA viruses. However, very little is known about RNA-DNA HR. Previously, it was demonstrated that synthetic RNA-containing molecules can serve as templates for repairing defective or broken homologous chromosomal DNA in yeast, human and bacterial cells, but it remained unclear whether cellular RNA transcripts can recombine with genomic DNA. Here, we investigated whether yeast cells can use transcript RNA as a template to repair a chromosomal DSB either directly or indirectly, if the RNA is converted first into a DNA copy, cDNA. We developed a system to detect HR between chromosomal DNA and transcript RNA in budding yeast, Saccharomyces cerevisiae. We focused on repair of a chromosomal DSB occurring either in a homologous but remote locus (trans) or in the same transcript-generating locus (cis) in yeast. We proved that transcript RNA can repair a DSB indirectly, via cDNA. Moreover, we found that cDNA repair is much more frequent in the trans than in the cis system. Interestingly, in the absence of Ribonuclease H1 and H2 (RNases H1 and H2), we could detect DSB repair even in conditions that strongly inhibit cDNA formation, suggesting direct DSB repair by transcript RNA. In contrast to DSB repair by cDNA, the direct DSB repair by transcript RNA is more efficient in the cis than in the trans system, despite the higher abundance of the transcript in the trans system. These results suggest that the vicinity of the transcript RNA to the break site in the cis system may facilitate DSB repair. DSB repair by transcript RNA in cis is promoted by the HR protein Rad52 but not Rad51, in agreement with the demonstration that the yeast and human Rad52 proteins efficiently catalyze annealing of RNA to a DSB-like DNA end in vitro. We also showed that yeast cells expressing hypomorphic mutants of RNase H2, which correspond to the human RNase H2 mutants that are associated with the neuroimmunological disease, Aicardi Goutieres (AGS) syndrome, have increased frequency of DSB repair by cDNA, significantly higher than in wild-type RNase H2 cells. In addition, we showed that in contrast to DSB repair by single strand DNA (ssDNA) oligonucleotides (oligos), RNA templated DSB repair is not dependent on factors that are major players in DNA end resection. This result could be explained by a mechanism in which transcript RNA repairs a DSB in conditions of limited end resection via an inverse strand exchange reaction. Our study provides proof and initial characterization of a new mechanism of DNA repair and HR mediated by RNA in yeast, and unravels novel aspects in the complex relationship between RNA and DNA in genome stability.Ph.D