Mismatch Tolerance During Homologous Recombination in Mammalian Cells

Abstract

We investigated the homology dependency of recombination in cultured thymidine kinase-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) which was rendered non-functional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a closely linked truncated “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via recombinational repair provoked by induction of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. We used three different donor sequences that contained 16, 25, or 33 mismatches relative to the recipient. The mismatches within these “homeologous” sequences were clustered and were surrounded by regions of high homology. Previous work indicated that mammalian cells fastidiously avoid recombination between homeologous sequences. We show that when homeologous sequences are surrounded by high homology, mismatches are frequently included in gene conversion events. Gene conversion tracts from spontaneous recombination revealed that in each event either all or none of the mismatches were transferred from donor to recipient, suggesting that recombination must begin and end in high homology. This requirement was somewhat relaxed for DSB-induced events. Knock-down of mismatch repair provided evidence that incorporation of mismatches into gene conversion tracts involved repair of mismatched heteroduplex intermediates. Our results indicate that mismatch repair of multiple mispaired bases does not function to impede exchange between homeologous sequences. Supported by grant MCB: 1157416 from the NSF to ASW and BCW.