Repair of DNA Double Strand Breaks by RNA/DNA Patches in U937 Cells.

Abstract 2375

It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we generated a vector containing the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk cDNA was the recognition site for the rare-cutting meganuclease I-SceI. This vector was electroporated into U937 cells, and a clone containing a single copy, of the EF1aTK vector, designated F5, was isolated. Transfection of the F5 cells with an I-SceI expression vector, followed by ganciclovir selection, identified clones that had lost expression of HsTK. We recovered no GCR with this approach; most of the GCV-resistant clones had partial or complete deletions of the EF1a promoter, the HsTK cDNA, or both. However we noted that ∼5–10% of repair elements involved insertions of DNA sequences derived from distant regions of the genome; the length of the inserted fragment varied from 47 to 756 bp. Surprisingly, all of the inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE (Long Interspersed Nuclear Element) or SINE (Short Interspersed Nuclear Element) 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 test the hypothesis that the patches at the DNA DSB sites could be derived from RNA. After hygromycin selection for successfully transfected cells, genomic DNA was isolated, and amplified with primers that flanked the I-SceI cleavage site. DNA fragments of 500–1000bp (larger than the size of the uncleaved EF!aTK PCR product, which was 400 bp) containing insertions at the I-SceI site were isolated, subcloned, and sequenced. We identified 51 independent sequences which had insertions of 23–266 bp at I-SceI cleavage site. Of these 51 insertions, 4 were vector capture events (derived form the expression vector), and 4 were too short to identify unambiguously. Of the remaining 43 insertions, 39 were derived from a single genomic loci, and 4 samples contained identifiable sequences from 2 distinct genomic regions. The sequences were derived from 16 of the 24 human chromosomes, with no clear preference for any specific chromosome. 62% of the sequences were found to contain sequences from a transcribed gene region, and 64% 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. Furthermore, 45 of the 47 inserted sequences were derived from endogenous human sequences; however 2 insertions matched to mouse sequences, and were derived from the co-transfected mouse RNA. One sample matched an intronic region of murine Vwa3b, which we confirmed was highly expressed in the mouse cells used to harvest the RNA used for the co-transfection. A second sample was not a gene sequence but contained a LINE element.

Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome. This is the first systematic study focused on captured sequences at DNA DSB sites in the mammalian genome, and suggests that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Finally, the observation that I-SceI-induced DNA DSBs are often repaired by insertions suggests that these insertions could be mistaken for chromosome translocations, if only one “side” of the DNA DSB is sequenced.