Role of Homology and DNA Double‐Strand Breaks in Initiation of Intrachromosomal Recombination in Mammalian Cells

Abstract

As part of our continuing investigations into the mechanisms of intrachromosomal recombination in mammalian cells, we stably transfected thymidine kinase deficient mouse fibroblasts with a DNA construct, pKM1, harboring a herpes thymidine kinase (tk) gene rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I‐SceI. We refer to this latter tk sequence as the “recipient.” The construct also contained a closely linked truncated “donor” tk sequence. The recipient and donor were positioned as direct repeats with respect to transcriptional orientation. The donor could potentially restore function to the recipient gene via recombination with the recipient, and such events could be provoked by induction of a double‐strand break (DSB) at the I‐SceI site in the recipient. Such DSB repair events were recoverable by selection for tk‐positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient in pKM1, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Our earlier work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In work presented here, we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest that the search for high homology prior to recombination is not mediated by an invading DNA terminus and may actually be independent of DSB formation. Further, the homology that enabled the recovered recombination events was located at a discrete distance from the actual invading DNA terminus. In an extension of our studies, we designed two additional constructs, pCW1 and pCW2. These constructs were similar to pKM1 except the recipient sequence in each was disrupted by an 8bp XhoI linker insertion and each construct contained a I‐SceI recognition site positioned within vector sequences at a location outside of either the recipient or donor sequence. Cells transfected with constructs pCW1 and pCW2 are being used to further explore whether a chromosomal DSB can induce and/or influence the nature of genetic exchanges occurring at a discrete distance from the DSB when the genetic exchanges themselves do not directly repair the DSB. Progress addressing this latter issue is presented.Support or Funding InformationThis work was supported by grant MCB 1157416 from the National Science Foundation to ASW and BCW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.