Abstract 639: Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR). To study DNA DSB repair in vivo, we generated a vector with the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk was the recognition site for the meganuclease I-SceI. The EF1aTk vector was introduced into U937 cells and a clone (designated F5) with a single copy of the vector was isolated. Transfection of F5 cells with an I-SceI expression vector, followed by ganciclovir (GCV) selection, identified clones that lost expression of HsTK. We recovered no GCR, as most of the GCV-resistant clones had deletions of the EF1a promoter or the HsTK. However ∼10% of the clones had insertions (47-756 bp) of DNA sequences derived from distant regions of the genome; all inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE or SINE sequences. Therefore, we hypothesized that the inserted sequences used to “patch” the DNA DSB could be based on reverse transcription of an RNA template. Since sequences derived from human RNA and human genomic DNA are identical (with the exception of RNA splice events, poly-A tails, and RNA-edited nucleotides), we co-transfected the F5 cells with murine RNA and an I-SceI expression vector to determine if patches at the DNA DSB sites could be derived from RNA. We identified 121 independent sequences which represented insertions of 20-609 bp at the I-SceI cleavage site. Of these 121 insertions, 6 were vector or E. coli capture events and 7 were too short to identify. The chromosomal origin of 5 insertions could not be assigned as these insertions consisted of 5 or 6 bp tandem repeat sequences; 3 had a (CCCTAA)n repeat, identical to a mammalian telomere repeat. Of the remaining 103 insertions, 94 were derived from a single genomic loci, and 9 samples contained sequences from more than 2 distinct genomic regions. The sequences were derived from 22 of the 24 human chromosomes. 65% of the sequences were found to contain sequences from a transcribed gene region, and 52% contained repeat sequences such as LINE, SINE, or LTR. The involvement of retrotransposon sequences, which are known to be reverse transcribed and integrated into the genome, supports the hypothesis that at least some of the insertions may be templated from RNA. Although most insertions were derived from human sequences; 9 insertions matched murine sequences, and were derived from the co-transfected mouse RNA. All of these were derived from gene and/or retrotransposon sequence. Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome, and suggest that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Citation Format: Masahiro Onozawa, Tamas Varga, Zhenhua Zhang, Yoo Jung Kim, Peter Aplan. Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 639. doi:10.1158/1538-7445.AM2013-639