Double-strand breaks (DSBs) are a common spontaneous or damage-induced lesion and are central to many directed, natural genetic changes. We created systems based on repair by oligonucleotides that provide unique opportunities to examine DSB repair and utilize the repair for rapid modification of the genome. The oligonucleotide targeting approach for genetic modification eliminated many of the steps required to produce multiple changes in the genome and was recently expanded to specifically address targeting to a DSB. This versatile and accurate system for in vivo targeted mutagenesis has been referred to as delitto perfetto since there is complete elimination of the marker sequences that are used for selection. In this way, no heterologous sequence remains and multiple rounds of mutagenesis can be accomplished by a self-cloning process applicable to any target sequence. We found that an oligonucleotide is capable of repairing a DSB, resulting in a new tool to address DSB repair. A novel cassette that could be targeted anywhere in the genome was created that contained a regulatable I-SceI endonuclease and an I-SceI double-strand cut site in order to produce a DSB at will in a region to be targeted by oligonucleotides. The DSB stimulated oligonucleotide targeting more than 1000-fold, with targeting efficiencies as high as 20% of all cells. A DSB could also stimulate recombination with single-strand DNA (ss-DNA), suggesting new twists on present models of DSB repair. The extremely high transformation frequencies and versatility of the break-mediated delitto perfetto system, has resulted in new powerful tools for dissecting mechanisms of homologous recombination as well as rapid genome modification from point mutations to chromosome rearrangements. We investigated the genetic requirements and found that ss-oligonucleotide-directed repair occurred exclusively via Rad52 and Rad59-mediated single-strand annealing (SSA). The repair did not involve Rad51-driven strand invasion. In fact, suppression of strand invasion increased oligonucleotide repair. Even the SSA domain (N-terminal) of human Rad52 provided partial complementation for a null rad52 mutation and the repair is similar to repair mediated by the homologous truncated yeast protein, in that it is independent of Rad51 and partially dependent on Rad59. Our results with human Rad52 containing the N-terminal provide the first direct evidence for hRad52 SSA activity in vivo. A DSB was shown not only to stimulate unbiased targeting of ss-oligonucleotides with homology to both sides of a DSB, but also to activate targeting by oligonucleotides homologous to only one side of the DSB at large (over 20 kb) distances from the DSB in a strand-biased manner. This suggests extensive 5 to 3 resection followed by restoration of resected DNA to the double-strand state. We concluded that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by ss-DNA can be conservative (error free) and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs. ? We also found that not only DNA but also RNA oligonucleotides can directly repair a chromosomal DSB in yeast in a homology driven manner. Precise repair was accomplished with RNA oligonucleotides that targeted a chromosomal change, showing that DNA synthesis occurred on the RNA template. It was long known that RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons and in the elongation of telomeres. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining3. An indirect role for RNA in DNA repair, following reverse transcription and formation of a cDNA, had been observed previously for the non-homologous joining of DSB ends. In the yeast Saccharomyces cerevisiae, where homologous recombination is efficient, RNA was shown to mediate recombination, but only indirectly via a cDNA intermediate generated by the reverse transcriptase (RT) function of Ty retrotransposons in the cytoplasm within the confines of Ty particles. While pairing between duplex DNA and single-strand (ss) RNA can occur in vitro and in vivo, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We showed that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. The in vivo results are supported by in vitro biochemistry, which establishes the feasibility of a key step that would be required for DSB repair by RNA. In collaboration with K. Bebenek and T. Kunkel in LMG, we showed that replicative DNA polymerases alpha and delta are able to synthesize DNA on RNA templates. Our study identifies a novel cellular mechanism for direct transfer of information from RNA templates to nuclear DNA during DSB repair. Based on this new RNA capability, we suggest that endogenous RNA could play a direct role in repairing lesions during or after transcription, especially given its high local concentration. Our results set the stage for understanding how direct, homology-driven transfer of endogenous RNA information to DNA may occur. These observations add a new chapter to the recently described functions ascribed to RNA that include silencing, translational control by micro RNAs, RNA-mediated epigenetic changes, generation of deletions. In addition to understanding how RNA as a homologous template may contribute to genomic DNA integrity and evolution, this study could open a new direction in gene targeting/therapy where RNA templates could be used for targeting genomic changes, especially since RNA can be amplified within the cell.
