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. ? Site-directed mutagenesis systems in which specific DNA sequences are targeted for alteration in vitro have been instrumental in dissecting genetic pathways, gene regulation and in understanding structure-function relationships in proteins. Often there is a need for direct in vivo modification. However, the modification of genomic DNA within cells, such that no heterologous material is retained, is generally an elaborate and inefficient process, that includes various cloning steps for each mutant allele that is to be created. We developed an oligonucleotide targeting approach that eliminated many of the steps required to produce multiple changes in the genome. Briefly, the first step involves integration of a COunterselectable REporter (CORE) cassette that is targeted to a desired genomic locus. The gene modification step occurs by transformation with the appropriate transforming oligonucleotides such that the CORE cassette is lost and the appropriate change is generated. This versatile and accurate system for in vivo targeted mutagenesis has been referred to as delitto perfetto [Italian for perfect murder and idiomatic for prefect deletion] 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 what might be considered as a self-cloning process applicable to any target sequence. The system has been applied extensively in many studies in and beyond our laboratory to rapidly generate mutants of yeast genes or genes from other organisms cloned in yeast. While developed in the yeast Saccharomyces cerevisiae where homologous recombination is highly efficient, the approach could be applied to other organisms.? ? Recently, we extended these findings to the repair of a DSB. We proposed that an oligonucleotide might be capable of repairing a DSB and thereby provide a new tool to address DSB repair. A DSB-CORE cassette was created that contained a regulatable I-SceI endonuclease and an I-SceI double-strand cut site within the original CORE cassette described above. A site-specific DSB could, therefore, be generated by the I-SceI just prior to oligonucleotide transformation. We found that the DSB stimulated oligonucleotide targeting more than 1000-fold, with targeting efficiencies as high as 20% of all cells. This is over 2 orders of magnitude higher than any reported DNA integration frequency in yeast. We also found that a DSB can strongly 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 gross chromosome rearrangements such as chromosome circularization, chromosome fusion and reciprocal translocations. ? In order to identify the processes involved in the efficient repair of a chromosomal DSB by ss-DNA we investigated the genetic requirements. We demonstrated 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 and, moreover, suppression of strand invasion increased repair with oligonucleotides. Even the SSA domain (N-terminal) of human Rad52 provided partial complementation for a null rad52 mutation. Moreover, we showed that this 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 (over20 kb) distances from the DSB in a strand-biased manner. These results suggest 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. The finding that resection is extensive is now be extended to studies on the genetic stability of single strand DNA in the genome and possible unique genetic sensitivity to spontaneous spontaneous and damage-induced changes (also part of ES065073-16). ? We also discovered 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. 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. Through a 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. Importantly, the presence of a 20-base DNA non-homologous tail at the 3?-end of the RNA oligonucleotides increased more than 100-fold the repair frequency by oligonucleotides containing homologous RNA suggesting that 3'-degradation is a major factor preventing efficient targeting with RNA. Our study identifies a novel cellular mechanism for direct transfer of information from RNA templates to nuclear DNA during DSB repair and may open new directions in gene targeting/therapy, since RNA molecules can be amplified at will within the cells.
